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  • 1.
    Apostolov, Rossen
    et al.
    KTH, Skolan för datavetenskap och kommunikation (CSC), Centra, Parallelldatorcentrum, PDC.
    Yonezawa, Yasushige
    Standley, Daron M
    Kikugawa, Gota
    Takano, Yu
    Nakamura, Haruki
    Membrane attachment facilitates ligand access to the active site in monoamine oxidase A2009Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, nr 25, s. 5864-5873Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Monoamine oxidase membrane enzymes are responsible for the catalytic breakdown of extra- and intracellular neurotransmitters and are targets for the development of central nervous system drugs. We analyzed the dynamics of rat MAOA by performing multiple independent molecular dynamics simulations of membrane-bound and membrane-free forms to clarify the relationship between the mechanics of the enzyme and its function, with particular emphasis on the significance of membrane attachment. Principal component analysis of the simulation trajectories as well as correlations in the fluctuations of the residues pointed to the existence of three domains that define the global dynamics of the protein. Interdomain anticorrelated movements in the membrane-bound system facilitated the relaxation of interactions between residues surrounding the substrate cavity and induced conformational changes which expanded the active site cavity and opened putative pathways for substrate uptake and product release. Such events were less pronounced in the membrane-free system due to differences in the nature of the dominant modes of motion. The presence of the lipid environment is suggested to assist in decoupling the interdomain motions, consistent with the observed reduction in enzyme activity under membrane-free conditions. Our results are also in accordance with mutational analysis which shows that modifications of interdomain hinge residues decrease the activity of rat MAOA in solution.

  • 2. Branneby, Cecilia
    et al.
    Carlqvist, Peter
    KTH, Skolan för kemivetenskap (CHE), Kemi, Fysikalisk kemi (stängd 20110630).
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi (stängd 20130101).
    Brinck, Tore
    KTH, Skolan för kemivetenskap (CHE), Kemi, Fysikalisk kemi (stängd 20110630).
    Berglund, Per
    KTH, Tidigare Institutioner, Bioteknologi.
    Rational redesign of a lipase to an aldolase2003Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 42, nr 28, s. 8633-8633Artikkel i tidsskrift (Fagfellevurdert)
  • 3. Busenlehner, Laura S.
    et al.
    Alander, Johan
    Jegerscohld, Caroline
    Holm, Peter J.
    Bhakat, Priyaranjan
    Hebert, Hans
    KTH, Skolan för teknik och hälsa (STH), Strukturell bioteknik.
    Morgenstern, Ralf
    Armstrong, Richard N.
    Location of substrate binding sites within the integral membrane protein microsomal glutathione transferase-12007Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, nr 10, s. 2812-2822Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Microsomal glutathione transferase-1 (MGST1) is a trimeric, membrane-bound enzyme with both glutathione (GSH) transferase and hydroperoxidase activities. As a member of the MAPEG superfamily, MGST1 aids in the detoxication of numerous xenobiotic substrates and in cellular protection from oxidative stress through the GSH-dependent reduction of phospholipid hydroperoxides. However, little is known about the location of the different substrate binding sites, including whether the transferase and peroxidase activities overlap structurally. Although molecular density attributed to GSH has been observed in the 3.2 A resolution electron crystallographic structure of MGST1, the electrophilic and phospholipid hydroperoxide substrate binding sites remain elusive. Amide H-D exchange kinetics and H-D ligand footprinting experiments indicate that GSH and hydrophobic substrates bind within similar, but distinct, regions of MGST1. Site-directed mutagenesis, guided by the H-D exchange results, demonstrates that specific residues within the GSH footprint effect transferase activity toward 1-chloro-2,4-dinitrobenzene. In addition, cytosolic residues surrounding the chemical stress sensor C49 but not modeled in the crystal structure appear to play an important role in the formation of the binding site for hydrophobic substrates. Although the fatty acid/phospholipid binding site structurally overlaps that for GSH, it does not appear to be localized to the same region as other hydrophobic substrates. Finally, H-D exchange mass spectrometry reveals a specific conformational transition that may mediate substrate binding and/or product release. Such structural changes in MGST1 are essential for activation of the enzyme and are important for its biological function.

  • 4. Busenlehner, L.S.
    et al.
    Codreanu, S.G.
    Holm, P.J.
    Bhakat, P.
    Hebert, Hans
    Karolinska Institutet.
    Morgenstern, R.
    Armstrong, R.N.
    Stress sensor triggers conformational response of the integral membrane protein microsomal glutathione transferase 12004Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, nr 35, s. 11145-11152Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Microsomal glutathione (GSH) transferase 1 (MGST1) is a trimeric, integral membrane protein involved in cellular response to chemical or oxidative stress. The cytosolic domain of MGST1 harbors the GSH binding site and a cysteine residue (C49) that acts as a sensor of oxidative and chemical stress. Spatially resolved changes in the kinetics of backbone amide H/D exchange reveal that the binding of a single molecule of GSH/trimer induces a cooperative conformational transition involving movements of the transmembrane helices and a reordering of the cytosolic domain. Alkylation of the stress sensor preorganizes the helices and facilitates the cooperative transition resulting in catalytic activation.

  • 5. Comfort, Donald A.
    et al.
    Bobrov, Kirill S.
    Ivanen, Dina R.
    Shabalin, Konstantin A.
    Harris, James M.
    Kulminskaya, Anna A.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Kelly, Robert M.
    Biochemical analysis of Thermotoga maritima GH36 alpha-galactosidase (TmGalA) confirms the mechanistic commonality of clan GH-D glycoside hydrolases2007Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, nr 11, s. 3319-3330Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Organization of glycoside hydrolase (GH) families into clans expands the utility of information on catalytic mechanisms of member enzymes. This issue was examined for GH27 and GH36 through biochemical analysis of GH36 alpha-galactosidase from Thermotoga maritima (TmGalA). Catalytic residues in TmGalA were inferred through structural homology with GH27 members to facilitate design of site-directed mutants. Product analysis confirmed that the wild type (WT) acted with retention of anomeric stereochemistry, analogous to GH27 enzymes. Conserved acidic residues were confirmed through kinetic analysis of D327G and D387G mutant enzymes, azide rescue, and determination of azide rescue products. Mutation of Asp327 to Gly resulted in a mutant that had a 200-800-fold lower catalytic rate on aryl galactosides relative to the WT enzyme. Azide rescue experiments using the D327G enzyme showed a 30-fold higher catalytic rate compared to without azide. Addition of azide to the reaction resulted in formation of azide beta-D-galactopyranoside, confirming Asp327 as the nucleophilic residue. The Asp387Gly mutation was 1500-fold catalytically slower than the WT enzyme on p-nitrophenyl alpha-D-galactopyranoside. Analysis at different pH values produced a bell-shaped curve of the WT enzyme, but D387G exhibited higher activity with increasing pH. Catalyzed reactions with the D387G mutant in the presence of azide resulted in formation of azide alpha-D-galactopryanoside as the product of a retaining mechanism. These results confirm that Asp387 is the acid/base residue of TmGalA. Furthermore, they show that the biochemical characteristics of GH36 TmGalA are closely related to GH27 enzymes, confirming the mechanistic commonality of clan GH-D members.

  • 6. Dahl, Göran
    et al.
    Gutierrez Arenas, Omar
    Department of Biochemistry and Organic Chemistry, Uppsala University.
    Danielson, U Helena
    Hepatitis C virus NS3 protease is activated by low concentrations of protease inhibitors2009Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, nr 48, s. 11592-11602Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) is a bifunctional enzyme with a protease and a helicase functionality located in each of the two domains of the single peptide chain. There is little experimental evidence for a functional role of this unexpected arrangement since artificial single domain forms of both enzymes are catalytically competent. We have observed that low concentrations of certain protease inhibitors activate the protease of full-length NS3 from HCV genotype 1a with up to 100%, depending on the preincubation time and the inhibitor used. The activation was reduced, but not eliminated, by increased ionic strength, lowered glycerol concentration, or lowered pH. In all cases, it was at the expense of a significant loss of activity. Activation was not seen with the artificial protease domain of genotype 1b NS3 fused with a fragment of the NS4A cofactor. This truncated and covalently modified enzyme form was much less active and exhibited fundamentally different catalytic properties to the full-length NS3 protease without the fused cofactor. The most plausible explanation for the activation was found to involve a slow transition between two enzyme conformations, which differed in their catalytic ability and affinity for inhibitors. Equations derived based on this assumption resulted in better fits to the experimental data than the equation for simple competitive inhibition. The mechanism may involve an inhibitor-induced stabilization of the helicase domain in a conformation that enhances the protease activity, or an improved alignment of the catalytic triad in the protease. The proposed mnemonic mechanism and derived equations are viable for both these explanations and can serve as a basic framework for future studies of enzymes activated by inhibitors or other ligands.

  • 7. Dimitroff, George
    et al.
    Little, Alan
    Lahnstein, Jelle
    Schwerdt, Julian G.
    Srivastava, Vaibhav
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Bulone, Vincent
    Burton, Rachel A.
    Fincher, Geoffrey B.
    (1,3;1,4)-beta-Glucan Biosynthesis by the CSLF6 Enzyme: Position and Flexibility of Catalytic Residues Influence Product Fine Structure2016Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 55, nr 13, s. 2054-2061Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Cellulose synthase-like F6 (CslF6) genes encode polysaccharide synthases responsible for (1,3;1,4)-beta-glucan biosynthesis in cereal grains. However, it is not clear how both (1,3)- and (1,4) -linkages are incorporated into a single polysaccharide chain and how the frequency and arrangement of the two linkage types that define the fine structure of the polysaccharide are controlled. Through transient expression in Nicotiana benthamiana leaves, two CSLF6 orthologs from different cereal species were shown to mediate the synthesis of (1,3;1,4)-beta-glucans with very different fine structures. Chimeric cDNA constructs with interchanged sections of the barley and sorghum CslF6 genes were developed to identify regions of the synthase enzyme responsible for these differences. A single amino acid residue upstream of the TED motif in the catalytic region was shown to dramatically change the fine structure of the polysaccharide produced. The structural basis of this effect can be rationalized by reference to a homology model of the enzyme and appears to be related to the position and flexibility of the TED motif in the active site of the enzyme. The region and amino acid residue identified provide opportunities to manipulate the solubility of (1,3;1,4)-beta-glucan in grains and vegetative tissues of the grasses and, in particular, to enhance the solubility of dietary fibers that are beneficial to human health.

  • 8. Hariharan, Parameswaran
    et al.
    Andersson, Magnus
    KTH, Skolan för teknikvetenskap (SCI), Teoretisk fysik, Beräkningsbiofysik. KTH, Centra, Science for Life Laboratory, SciLifeLab.
    Jiang, Xiaoxu
    Pardon, Els
    Steyaert, Jan
    Kaback, H. Ronald
    Guan, Lan
    Thermodynamics of Nanobody Binding to Lactose Permease2016Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 55, nr 42, s. 5917-5926Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Camelid nanobodies (Nbs) raised against the outward-facing conformer of a double-Trp mutant of the lactose permease of Escherichia coli (LacY) stabilize the permease in outward-facing conformations. Isothermal titration calorimetry is applied herein to dissect the binding thermodynamics of two Nbs, one that markedly improves access to the sugar-binding site and another that dramatically increases the affinity for galactoside. The findings presented here show that both enthalpy and entropy contribute favorably to binding of the Nbs to wild-type (WT) LacY and that binding of Nb to double-Trp mutant G46W/G262W is driven by a greater enthalpy at an entropic penalty. Thermodynamic analyses support the interpretation that WT LacY is stabilized in outward-facing conformations like the double-Trp mutant with closure of the cytoplasmic cavity through conformational selection. The LacY conformational transition required for ligand binding is reflected by a favorable entropy increase. Molecular dynamics simulations further suggest that the entropy increase likely stems from release of immobilized water molecules primarily from the cytoplasmic cavity upon closure.

  • 9. Hart, D. O.
    et al.
    He, S. M.
    Chany, C. J.
    Withers, S. G.
    Sims, P. F. G.
    Sinnott, M. L.
    Brumer, Harry
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Identification of Asp-130 as the catalytic nucleophile in the main alpha-galactosidase from Phanerochaete chrysosporium, a family 27 glycosyl hydrolase2000Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 39, nr 32, s. 9826-9836Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Characterization of the complete gene sequence encoding the alpha-galactosidase from Phanerochaete chrysosporium confirms that this enzyme is a member of glycosyl hydrolase family 27 [Henrissat, B., and Bairoch, A. (1996) Biochem. J. 316, 695-696]. This family, together with the family 36 alpha-galactosidases, forms glycosyl hydrolase dan GH-D, a superfamily of alpha-galactosidases, alpha-N-acetylgalactosaminidases, and isomaltodextranases which are likely to share a common catalytic mechanism and structural topology. Identification of the active site catalytic nucleophile was achieved by labeling with the mechanism-based inactivator 2',4',6'-trinitrophenyl 2-deoxy-2,2-difluoro-alpha-D- lyxo-hexopyranoside; this inactivator was synthesized by anomeric deprotection of the known 1,3,4,6-tetra-O-acetyl-2-deoxy-2,2-difluoro-D-lyxo-hexopyranoside [McCarter, J. D., Adam, M. J., Braun, C., Namchuk, M., Tull, D., and Withers, S. G. (1993) Carbohydr. Res. 249, 77-90], picrylation with picryl fluoride and 2,6-di-tert-butylpyridine, and O-deacetylation with methanolic HCl. Enzyme inactivation is a result of the formation of a stable 2-deoxy-2,2-difluoro-beta-D-lyxo-hexopyranosyl-enzyme intermediate. Following peptic digestion, comparative liquid chromatographic/mass spectrometric analysis of inactivated and control enzyme samples served to identify the covalently modified peptide. After purification of the labeled peptide, benzylamine was shown to successfully replace the 2-deoxy-2,2-difluoro-D-lyxo-hexopyranosyl peptidyl ester by aminolysis. The labeled amino acid was identified as Asp-130 of the mature protein by further tandem mass spectrometric analysis of the native and derivatized peptides in combination with Edman degradation analysis. Asp-130 is found within the sequence YLKYDNC, which is highly conserved in all known family 27 glycosyl hydrolases.

  • 10.
    Hedin, Eva . M. K.
    et al.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Hoyrup, P.
    Patkar, S. A.
    Vind, J.
    Svendsen, A.
    Fransson, L.inda
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Hult, Karl
    KTH, Tidigare Institutioner                               , Biokemi och biokemisk teknologi.
    Interfacial orientation of Thermomyces lanuginosa lipase on phospholipid vesicles investigated by electron spin resonance relaxation spectroscopy2002Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, nr 48, s. 14185-14196Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The binding orientation of the interfacially activated Thermomyces lanuginosa lipase (TLL, EC 3.1.1.3) on phospholipid vesicles was investigated using site-directed spin labeling and electron spin resonance (ESR) relaxation spectroscopy. Eleven TLL single-cysteine mutants, each with the mutation positioned at the surface of the enzyme, were selectively spin labeled with the nitroxide reagent (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl) methanethiosulfonate. These were studied together with small unilamellar vesicles (SUV) consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), to which TLL has previously been shown to bind in a catalytically active form [Cajal, Y., et al. (2000) Biochemistry, 39, 413-423]. The orientation of TLL with respect to the lipid membrane was investigated using, a water-soluble spin relaxation agent. chromium(III) oxalate (Crox), and a recently developed ESR relaxation technique [Lin, Y., et al. (1998) Science 279, 1925-1929], here modified to low microwave amplitude (< 0.36 G). The exposure to Crox for the spin label at the different positions on the surface of TLL was determined in the absence and presence of vesicles. The spin label at positions Gly61-Cys and Thr267-Cys, closest to the active site nucleophile Ser146 of the positions analyzed, displayed the lowest exposure factors to the membrane-impermeable spin relaxant, indicating the proximity to the vesicle surface. As an independent technique, fluorescence spectroscopy was employed to measure fluorescence quenching of dansyl-labeled POPG vesicles as exerted by the protein-bound spin labels. The resulting Stern-Volmer quenching constants showed excellent agreement with the ESR exposure factors. An interfacial orientation of TLL is proposed on the basis of the obtained results.

  • 11.
    Hedin, Eva M. K.
    et al.
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Hoyrup, P.
    Patkar, S. A.
    Vind, J.
    Svendsen, A.
    Hult, Karl
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Implications of surface charge and curvature for the binding orientation of Thermomyces lanuginosus lipase on negatively charged or zwitterionic phospholipid vesicles as studied by ESR spectroscopy2005Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, nr 50, s. 16658-16671Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The triglyceride lipase (EC 3.1.1.3) Thermomyces lanuginosus lipase (TLL) binds with high affinity to unilamellar phospholipid vesicles that serve as a diluent interface for both lipase and substrate, but it displays interfacial activation on only small and negatively charged such vesicles [Cajal, Y., et al. (2000) Biochemistry 39, 413-423]. The productive-mode binding orientation of TLL at the lipid-water interface of small unilamellar vesicles (SUV) consisting of 1-palmitoyl-2-oleoyi-sn-glycero-3-phosphati-dylglycerol (POPG) was previously determined using electron spin resonance (ESR) spectroscopy in combination with site-directed spin-labeling [Hedin, E. M. K., et al. (2002) Biochemistry 41, 1418514196]. In our investigation, we have studied the interfacial orientation of TLL when bound to large unilamellar vesicles (LUV) consisting of POPG, and bound to SUV consisting of 1-palmitoyl-2-oleoylsn-glycero-3-phosphatidylcholine (POPC). Eleven single-cysteine TLL mutants were spin-labeled as previously described, and studied upon membrane binding using the water soluble spin-relaxation agent chromium(III) oxalate (Crox). Furthermore, dansyl-labeled vesicles revealed the intermolecular fluorescence quenching efficiency between each spin-label positioned on TLL, and the lipid membrane. ESR exposure and fluorescence quenching data show that TILL associates closer to the negatively charged PG surface than the zwitterionic PC surface, and binds to both POPG LUV and POPC SUV predominantly through the concave backside of TLL opposite the active site, as revealed by the contact residues K74C-SL, R209C-SL, and T192C-SL. This orientation is significantly different compared to that on the POPG SUV, and might explain the differences in activation of the lipase. Evidently, both the charge and accessibility (curvature) of the vesicle surface determine the TLL orientation at the phospholipid interface.

  • 12.
    Hober, Sophia
    et al.
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Forsberg, G
    Palm, G
    Hartmanis, M
    Nilsson, B
    Disulfide exchange folding of insulin-like growth factor I.1992Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 31, nr 6Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The disulfide exchange folding properties of insulin-like growth factor I (IGF-I) have been analyzed in a redox buffer containing reduced (10 mM) and oxidized (1 mM) glutathione. Under these conditions, the 3 disulfide bridges of the 70 amino acid peptide were not quantitatively formed. Instead, five major forms of IGF-I were detected, and these components were concluded to be in equilibrium as their relative amounts were similar starting from either reduced, native, or a mismatched variant of IGF-I containing two non-native disulfides. The different components in the mixtures were trapped by thiol alkylation using vinylpyridine and subsequently isolated by reverse-phase HPLC. The purified variants were further characterized using plasma desorption mass spectrometry and peptide mapping. Two of the five different forms were identified as native and mismatched IGF-I. One form was a variant with only one disulfide bond, and the other two major components had two disulfides formed. In a separate experiment, early refolding intermediates were trapped by pyridylethylation after only 90 s of refolding in the glutathione buffer, starting from reduced IGF-I. The intermediates were identical to the components observed at equilibrium, but at different relative concentrations. On the basis of the disulfide bond patterns of the different components in the equilibrium mixtures, we conclude that the disulfide between cysteines-47 and -52 in IGF-I is an unfavorable high-energy bond that may exist in the native molecule in a strained configuration.

  • 13.
    Hober, Sophia
    et al.
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Hansson, A
    Uhlén, Mathias
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Nilsson, B
    Folding of insulin-like growth factor I is thermodynamically controlled by insulin-like growth factor binding protein.1994Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 33, nr 22Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Insulin-like growth factor I (IGF-I) is thermodynamically unable to quantitatively form its native disulfides under reversible redox conditions in vitro [Hober et al. (1992) Biochemistry 31, 1749-1756]. These results prompted the question of how IGF-I may overcome this energetic problem in its folding in vivo. Here, we report that an IGF-I precursor, IGF-I-Ea, shows disulfide-exchange folding properties similar to those of mature IGF-I and, thus, is concluded not to overcome the identified folding problem of mature IGF-I. However, correct disulfide bonds are formed very efficiently when insulin-like growth factor binding protein 1 is added in equimolar amounts to IGF-I to the refolding mixture. On the basis of these results, we propose that one important function of at least one of the six homologous insulin-like growth factor binding proteins is to assist in the formation and maintenance of the native disulfides of IGF-I. To our knowledge, this is the first example where the folding of a mammalian protein or peptide in circulation has been demonstrated to be thermodynamically controlled by its binding protein. Speculatively, this could provide a mechanism to regulate the half-life of IGF-I in vivo by altering the interaction with insulin-like growth factor binding proteins.

  • 14.
    Hopmann, Kathrin H.
    et al.
    KTH, Skolan för bioteknologi (BIO), Teoretisk kemi.
    Himo, Fahmi
    KTH, Skolan för bioteknologi (BIO), Teoretisk kemi.
    Cyanolysis and Azidolysis of Epoxides by Haloalcohol Dehalogenase: Theoretical Study of the Reaction Mechanism and Origins of Regioselectivity2008Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, nr 17, s. 4973-4982Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Haloalcohol dehalogenase HheC catalyzes the reversible dehalogenation of vicinal haloalcohols to form epoxides and free halides. In addition, HheC is able to catalyze the irreversible and highly regioselective ring-opening of epoxides with nonhalide nucleophiles, such as CN- and N-3(-). For azidolysis of aromatic epoxides, the regioselectivity observed with HheC is opposite to the regioselectivity of the nonenzymatic epoxide-opening. This, together with a relatively broad substrate specificity, makes HheC a promising tool for biocatalytic applications. We have designed large quantum chemical models of the HheC active site and used density functional theory to study the reaction mechanism of the HheC-catalyzed ring-opening of (R)-styrene oxide with the nucleophiles CN- and N3-. Both the cyanolysis and the azidolysis reactions are shown to take place in a single concerted step. The results support the suggested role of the putative Ser132-Tyr145-Arg149 catalytic triad, where Tyr145 acts as a general acid, donating a proton to the substrate, and Arg149 interacts with Tyr145 and facilitates proton abstraction, while Ser132 positions the substrate and reduces the barrier for epoxide opening through interaction with the emerging oxyanion of the substrate. We have also studied the regioselectivity of (R)-styrene oxide opening for both the cyanolysis and the azidolysis reactions. The employed active site model was shown to be able to reproduce the experimentally observed beta-regioselectivity of HheC. In silico mutations of various groups in the HheC active site model were performed to elucidate the important factors governing the regioselectivity.

  • 15.
    Hsieh, Yves S. Y.
    et al.
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. Univ Adelaide, Australia.
    Zhang, Qisen
    Yap, Kuok
    Shirley, Neil J.
    Lahnstein, Jelle
    Nelson, Clark J.
    Burton, Rachel A.
    Millar, A. Harvey
    Bulone, Vincent
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap. Univ Adelaide, Australia.
    Fincher, Geoffrey B.
    Genetics, Transcriptional Profiles, and Catalytic Properties of the UDP-Arabinose Mutase Family from Barley2016Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 55, nr 2, s. 322-334Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Four members of the UDP-Ara mutase (UAM) gene family from barley have been isolated and characterized, and their map positions on chromosomes 2H, 3H, and 4H have been defined. When the genes are expressed in Escherichia coli, the corresponding HvUAM1, HvUAM2, and HvUAM3 proteins exhibit UAM activity, and the kinetic properties of the enzymes have been determined, including K-m, K-cat, and catalytic efficiencies. However, the expressed HvUAM4 protein shows no mutase activity against UDP-Ara or against a broad range of other nucleotide sugars and related molecules. The enzymic data indicate therefore that the HvUAM4 protein may not be a mutase. However, the HvUAM4 gene is transcribed at high levels in all the barley tissues examined, and its transcript abundance is correlated with transcript levels for other genes involved in cell wall biosynthesis. The UDP-L-Arap -> UDP-L-Araf reaction, which is essential for the generation of the UDP-Araf substrate for arabinoxylan, arabinogalactan protein, and pectic polysaccharide biosynthesis, is thermodynamically unfavorable and has an equilibrium constant of 0.02. Nevertheless, the incorporation of Araf residues into nascent polysaccharides clearly occurs at biologically appropriate rates. The characterization of the HvUAM genes opens the way for the manipulation of both the amounts and fine structures of heteroxylans in cereals, grasses, and other crop plants, with a view toward enhancing their value in human health and nutrition, and in renewable biofuel production.

  • 16. Ibatullin, Farid M.
    et al.
    Baumann, Martin J.
    Greffe, Lionel
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Kinetic analyses of retaining endo-(xylo)glucanases from plant and microbial sources using new chromogenic xylogluco-oligosaccharide aryl glycosides2008Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, nr 29, s. 7762-7769Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A library of phenyl beta-glycosides of xylogluco-oligosaccharides was synthesized via a chemoenzymatic approach to produce new, specific substrates for xyloglucanases. Tamarind xyloglucan was completely hydrolyzed to four, variably galactosylated component oligosaccharides based on GlC(4) backbones, using a Trichoderma endo-glucanase mixture. Oligosaccharide complexity could be further reduced by beta-galactosidase treament. Subsequent per-O-acetylation, alpha-bromination, phase-transfer glycosylation, and Zemplen deprotection yielded phenyl glycosides of XXXG and XLLG oligosaccharides with a broad range of aglycon pK(a) values. Kinetic and product analysis of the action of the archetypal plant endo-xyloglucanase, Tropaeolum majus NXG1, on these compounds indicated that formation of the glycosyl-enzyme intermediate was rate-limiting in the case of phenol leaving groups with pK(a) values of >7, leading exclusively to substrate hydrolysis. Conversely, substrates with aglycon pK(a) values of 5.4 gave rise to a significant amount of transglycosylation products, indicating a change in the relative rates of formation and breakdown of the glycosyl-enzyme, intermediate for these faster substrates. Notably, comparison of the initial rates of XXXG-Ar and XLLG-Ar conversion indicated that catalysis by TmNXG1 was essentially insensitive to the presence of galactose in the negative subsites for all leaving groups. More broadly, analysis of a selection of enzymes from CAZy families GH 5, 12, and 16 indicated that the phenyl glycosides are substrates for anomeric configuration-retaining endo-xyloglucanases but are not substrates for strict xyloglucan endo-transglycosylases (XETs). The relative activities of the GH 5, 12, and 16 endo-xyloglucanases toward GGGG-CNP, XXXG-CNP, and XLLG-CNP reflected those observed using analogous high molar mass polysaccharides. These new chromogenic substrates may thus find wide application in the discovery, screening, and detailed kinetic analysis of new xyloglucan-active enzymes.

  • 17. Irani, Mehdi
    et al.
    Törnvall, Ulrika
    Genheden, Samuel
    Larsen, Marianne Wittrup
    KTH, Skolan för bioteknologi (BIO), Biokemi.
    Hatti-Kaul, Rajni
    Ryde, Ulf
    Amino Acid Oxidation of Candida antarctica Lipase B Studied by Molecular Dynamics Simulations and Site-Directed Mutagenesis2013Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, nr 7, s. 1280-1289Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Molecular dynamics simulations have been performed on lipase B from Candida antarctica (CalB) in its native form and with one or two oxidized residues, either methionine oxidized to methionine sulfoxide, tryptophan oxidized to 5-hydroxytryptophan, or cystine oxidized to a pair of cysteic acid residues. We have analyzed how these oxidations affect the general structure of the protein as well as the local structure around the oxidized amino acid and the active site. The results indicate that the methionine and tryptophan oxidations led to rather restricted changes in the structure, whereas the oxidation of cystines, which also caused cleavage of the cystine S-S linkage, gave rise to larger changes in the protein structure. Only two oxidized residues caused significant changes in the structure of the active site, viz., those of the Cys-22/64 and Cys-216/258 pairs. Site-directed mutagenesis studies were also performed. Two variants showed a behavior similar to that of native CalB,(M83I and M129L), whereas W155Q and M72S had severely decreased specific activity. M83I had a slightly higher thermostability than native CalB. No significant increase in stability toward hydrogen peroxide was observed. The same mutants were also studied by molecular dynamics. Even though no significant increase in stability toward hydrogen peroxide was observed, the results from simulations and site-directed mutagenesis give some clues about the direction of further work on stabilization.

  • 18.
    Jansson, Magnus
    et al.
    KTH, Tidigare Institutioner, Biokemi och biokemisk teknologi.
    Uhlén, M
    Nilsson, B
    Structural changes in insulin-like growth factor (IGF) I mutant proteins affecting binding kinetic rates to IGF binding protein 1 and IGF-I receptor.1997Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 36, nr 14, s. 4108-4117Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Ligand binding properties of five single amino acid substituted variants (V11A, D12A, Q15A, Q15E, and F16A) of human insulin-like growth factor I (IGF-I) were analyzed with respect to their binding affinities and binding kinetics to recombinant IGF binding protein 1 (IGFBP-1) and a soluble form of the IGF type I receptor (sIGF-I(R)), respectively. Side chains of the substituted residues are all predicted to be the most surface exposed in the alpha-helical portion of the B-region of the IGF-I molecule. The IGF-I variants were produced as fusion proteins to a IgG(Fc) binding protein domain, Z. Ligand binding kinetic rates were determined using BIAcore biosensor interaction analysis technology. All IGF-I variants showed altered binding affinities to both IGFBP- I and sIGF-I(R). Secondary structure content of the IGF-I variants was estimated using far-UV circular dichroism spectroscopy, followed by variable selection secondary structure calculations. The amount of calculated alpha-helicity is reduced for all the mutants, most predominantly for IGF-I(V11A) and IGF-I(F16A) proteins. Surprisingly, most of the effects of reduced binding affinities to both target proteins are attributed to lowered on-rates of binding, and these are correlated with the amount of alpha-helicity in each IGF-I variant. In addition, in some of the IGF-I variants, lowered off-rates of binding are observed. From the results, we propose that IGF-I is unusually sensitive to structural changes by surface amino acid substitutions in the B-region of the molecule. Therefore, biochemical or biological properties of amino acid substituted variants of IGF-I cannot be used in a straightforward way to dissect the direct involvement in binding of individual amino acid residues since structural changes may be involved.

  • 19. Jiang, X.
    et al.
    Andersson, Magnus
    KTH, Skolan för teknikvetenskap (SCI), Teoretisk fysik.
    Chau, B. T.
    Wong, L. Y.
    Villafuerte, M. K. R.
    Kaback, H. R.
    Role of Conserved Gly-Gly Pairs on the Periplasmic Side of LacY2016Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 55, nr 31, s. 4326-4332Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    On the periplasmic side of LacY, two conserved Gly-Gly pairs in helices II and XI (Gly46 and Gly370, respectively) and helices V and VIII (Gly159 and Gly262, respectively) allow close packing of each helix pair in the outward (periplasmic)-closed conformation. Previous studies demonstrate that replacing one Gly residue in each Gly-Gly pair with Trp leads to opening of the periplasmic cavity with abrogation of transport activity, but an increased rate of galactoside binding. To further investigate the role of the Gly-Gly pairs, 11 double-replacement mutants were constructed for each pair at positions 46 (helix II) and 262 (helix VIII). Replacement with Ala or Ser results in decreased but significant transport activity, while replacements with Thr, Val, Leu, Asn, Gln, Tyr, Trp, Glu, or Lys exhibit very little or no transport. Remarkably, however, the double mutants bind galactoside with affinities 10-20-fold higher than that of the pseudo-WT or WT LacY. Moreover, site-directed alkylation of a periplasmic Cys replacement indicates that the periplasmic cavity becomes readily accessible in the double-replacement mutants. Molecular dynamics simulations with the WT and double-Leu mutant in the inward-open/outward-closed conformation provide support for this interpretation. 

  • 20. Jiang, X.
    et al.
    Villafuerte, M. K. R.
    Andersson, Magnus
    University of California at Irvine, United States.
    White, S. H.
    Kaback, H. R.
    Galactoside-binding site in LacY2014Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 53, nr 9, s. 1536-1543Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Although an X-ray crystal structure of lactose permease (LacY) has been presented with bound galactopyranoside, neither the sugar nor the residues ligating the sugar can be identified with precision at ∼3.5 Å. Therefore, additional evidence is important for identifying side chains likely to be involved in binding. On the basis of a clue from site-directed alkylation suggesting that Asn272, Gly268, and Val264 on one face of helix VIII might participate in galactoside binding, molecular dynamics simulations were conducted initially. The simulations indicate that Asn272 (helix VIII) is sufficiently close to the galactopyranosyl ring of a docked lactose analogue to play an important role in binding, the backbone at Gly268 may be involved, and Val264 does not interact with the bound sugar. When the three side chains are subjected to site-directed mutagenesis, with the sole exception of mutant Asn272 → Gln, various other replacements for Asn272 either markedly decrease affinity for the substrate (i.e., high KD) or abolish binding altogether. However, mutant Gly268 → Ala exhibits a moderate 8-fold decrease in affinity, and binding by mutant Val264 → Ala is affected only minimally. Thus, Asn272 and possibly Gly268 may comprise additional components of the galactoside-binding site in LacY.

  • 21.
    Jonsson Rudsander, Ulla
    et al.
    KTH, Skolan för bioteknologi (BIO), Centra, Strategiskt Centrum för Biomimetiska Material, BioMime.
    Sandstrom, Corine
    Piens, Kathleen
    KTH, Skolan för bioteknologi (BIO), Centra, Strategiskt Centrum för Biomimetiska Material, BioMime.
    Master, Emma
    KTH, Skolan för bioteknologi (BIO), Centra, Strategiskt Centrum för Biomimetiska Material, BioMime.
    Wilson, David B.
    Brumer, Harry
    KTH, Skolan för bioteknologi (BIO), Centra, Strategiskt Centrum för Biomimetiska Material, BioMime. KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Kenne, Lennart
    Teeri, Tuula T.
    KTH, Skolan för bioteknologi (BIO), Centra, Strategiskt Centrum för Biomimetiska Material, BioMime. KTH, Skolan för bioteknologi (BIO), Glykovetenskap.
    Comparative NMR analysis of cellooligosaccharide hydrolysis by GH9 bacterial and plant endo-1,4-ss-glucanases2008Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, nr 18, s. 5235-5241Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    H-1 NMR spectroscopy has been used to analyze the product profiles arising from the hydrolysis of cellooligosaccharides by family GH9 cellulases. The product profiles obtained with the wild type and several active site mutants of a bacterial processive endoglucanase, Tf Cel9A, were compared with those obtained by a randomly acting plant endoglucanase, PttCe19A. PttCe19A is an orthologue of the Arabidopsis endocellulase, Korrigan, which is required for efficient cellulose biosynthesis. As expected, poplar PttCe19A was shown to catalyze the degradation of cellooligosaccharides by inversion of the configuration of the anomeric carbon. The product analyses showed that the number of interactions between the glucose units of the substrate and the aromatic residues in the enzyme active sites determines the point of cleavage in both enzymes.

  • 22.
    Kim, Jungwook
    et al.
    Albert Einstein Coll Med.
    Tsai, Ping-Chuan
    Texas A&M Univ, Dept Chem.
    Chen, Shilu
    KTH, Skolan för bioteknologi (BIO), Teoretisk kemi.
    Himo, Fahmi
    KTH, Skolan för bioteknologi (BIO), Teoretisk kemi.
    Almo, Steven C.
    Albert Einstein Coll Med.
    Raushel, Frank M.
    Texas A&M Univ, Dept Chem.
    Structure of Diethyl Phosphate Bound to the Binuclear Metal Center of Phosphotriesterase2008Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, nr 36, s. 9497-9504Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The bacterial phosphotriesterase (PTE) from Pseudomonas diminuta catalyzes the hydrolysis of organophosphate esters at rates close to the diffusion limit. X-ray diffraction studies have shown that a binuclear metal center is positioned in the active site of PTE and that this complex is responsible for the activation of the nucleophilic water from solvent. In this paper, the three-dimensional structure of PTE was determined in the presence of the hydrolysis product, diethyl phosphate (DEP), and a product analogue, cacodylate. In the structure of the PTE−diethyl phosphate complex, the DEP product is found symmetrically bridging the two divalent cations. The DEP displaces the hydroxide from solvent that normally bridges the two divalent cations in structures determined in the presence or absence of substrate analogues. One of the phosphoryl oxygen atoms in the PTE−DEP complex is 2.0 Å from the α-metal ion, while the other oxygen is 2.2 Å from the β-metal ion. The two metal ions are separated by a distance of 4.0 Å. A similar structure is observed in the presence of cacodylate. Analogous complexes have previously been observed for the product complexes of isoaspartyl dipeptidase, d-aminoacylase, and dihydroorotase from the amidohydrolase superfamily of enzymes. The experimentally determined structure of the PTE−diethyl phosphate product complex is inconsistent with a recent proposal based upon quantum mechanical/molecular mechanical simulations which postulated the formation of an asymmetrical product complex bound exclusively to the β-metal ion with a metal−metal separation of 5.3 Å. This structure is also inconsistent with a chemical mechanism for substrate hydrolysis that utilizes the bridging hydroxide as a base to abstract a proton from a water molecule loosely associated with the α-metal ion. Density functional theory (DFT) calculations support a reaction mechanism that utilizes the bridging hydroxide as the direct nucleophile in the hydrolysis of organophosphate esters by PTE.

  • 23.
    Linda, Öjemyr
    et al.
    Stockholm University, dept Biochem and Biophys.
    Sandén, Tor
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Experimentell biomolekylär fysik.
    Widengren, Jerker
    KTH, Skolan för teknikvetenskap (SCI), Tillämpad fysik, Experimentell biomolekylär fysik.
    Brzezinski, Peter
    Stockholm University, dept Biochem and Biophys.
    Lateral Proton Transfer between the Membrane and a Membrane Protein2009Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, nr 10, s. 2173-2179Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Proton transport across biological membranes is a key step of the energy conservation machinery in living organisms, and it has been proposed that the membrane itself plays an important role in this process. In the present study we have investigated the effect of incorporation of a proton transporter, cytochrome c oxidase, into a membrane on the protonation kinetics of a fluorescent pH-sensitive probe attached at the surface of the protein. The results show that proton transfer to the probe was slightly accelerated upon attachment at the protein surface (similar to 7 x 10(10) s(-1) M-1, compared to the expected value of (1-2) x 10(10) s(-1) M-1), which is presumably due to the presence of acidic/His groups in the vicinity. Upon incorporation of the protein into small unilamellar phospholipid vesicles the rate increased by more than a factor of 400 to similar to 3 x 10(13) s(-1) M-1, which indicates that the protein-attached probe is in rapid protonic contact with the membrane surface. The results indicate that. the membrane acts to accelerate proton uptake by the membrane-bound proton transporter.

  • 24.
    Master, Emma
    et al.
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Rudsander, Ulla
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Zhou, Welin
    Henriksson, Hongbin
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Divne, Christina
    KTH, Tidigare Institutioner (före 2005), Bioteknologi. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Industriell bioteknologi.
    Denman, Stuart
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Wilson, David
    Teeri, Tuula
    KTH, Tidigare Institutioner (före 2005), Bioteknologi.
    Recombinant Expression and Enzymatic characterization of PttCel9A, a KOR homologue from Populus tremula x tremuloides2004Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, nr 31, s. 10080-10089Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    PttCel9A is a membrane-bound, family 9 glycosyl hydrolase from Populus tremula x tremuloides that is upregulated during secondary cell wall synthesis. The catalytic domain of PttCel9A, Delta(1-105)PttCel9A, was purified, and its activity was compared to TfCel9A and TfCel9B from Thermobifida fusca. Since aromatic amino acids involved in substrate binding at subsites -4, -3, and -2 are missing in PttCel9A, the activity of TfCel9A mutant enzymes W256S, W209A, and W313G was also investigated. Delta(1-105)PttCel9A hydrolyzed a comparatively narrow range of polymeric substrates, and the preferred substrate was (carboxymethyl)cellulose 4M. Moreover, Delta(1-105)PttCel9A did not hydrolyze oligosaccharides shorter than cellopentaose, whereas TfCel9A and TfCel9B hydrolyzed cellotetraose and cellotriose, respectively. These data suggest that the preferred substrates of PttCel9A are long, low-substituted, soluble cellulosic polymers. At 30degreesC and pH 6.0, the k(cat) for cellohexaose of Delta(1-105)PttCel9A, TfCel9A, and TfCel9B were 0.023 +/- 0.001, 16.9 +/- 2.0, and 1.3 +/- 0.2, respectively. The catalytic efficiency (k(cat)/K-m) of TfCel9B was 39% of that of TfCel9A, whereas the catalytic efficiency of Delta(1-105)PttCel9A was 0.04% of that of TfCel9A. Removing tryptophan residues at subsites -4, -3, and -2 decreased the efficiency of cellohexaose hydrolysis by TfCel9A. Mutation of W313 to G had the most drastic effect, producing a mutant enzyme with 1% of the catalytic efficiency of TfCel9A. The apparent narrow substrate range and catalytic efficiency of PttCel9A are correlated with a lack of aromatic amino acids in the substrate binding cleft and may be necessary to prevent excessive hydrolysis of cell wall polysaccharides during cell wall formation.

  • 25. Pelosi, L.
    et al.
    Imai, T.
    Chanzy, H.
    Heux, L.
    Buhler, E.
    Bulone, Vincent
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Structural and morphological diversity of (1 -> 3)-beta-D-glucans synthesized in vitro by enzymes from Saprolegnia monoica. Comparison with a corresponding in vitro product from blackberry (Rubus fruticosus)2003Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 42, nr 20, s. 6264-6274Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Detergent extracts of microsomal fractions from Saprolegnia monoica and blackberry (Rubus fruticosus) cells were incubated with UDP-glucose to yield in vitro (1-->3)-beta-D-glucans. The insoluble products were analyzed by conventional and cryo transmission electron microscopy, X-ray diffraction, and C-13 CP/MAS NMR, and their molecular weights were determined by light scattering experiments. All the products were microfibrillar, but for the detergent extracts from S. monoica, important morphological differences were observed when the pH of the synthesizing medium was modified. At pH 6, the product had a weight average degree of polymerization ((DPw) over bar) exceeding 20 000 and consisted of endless ribbon-like microfibrils. The microfibrils obtained at pH 9 had a length of only 200-300 nm, and their (DPw) over bar was similar to5000. Of all the in vitro (1-->3)-beta-D-glucans, the one from R. fruticosus had the shortest length and the smallest (DPw) over bar. Crystallographic and spectroscopic data showed that the three in vitro samples consisted of triple helices of (1-->3)-beta-D-glucans and contained substantial amounts of water molecules in their structure, the shortest microfibrils being more hydrated. In addition, the long microfibrils from S. monoica synthesized at pH 6 were more resistant toward the action of an endo-(1-->3)-beta-D-glucanase than the shorter ones obtained at pH 9. These results are discussed in terms of molecular biosynthetic mechanisms of fungal and plant (1-->3)-beta-D-glucans, and in relation with the possible existence of several (1-->3)-beta-D-glucan synthases in a given organism. The interpretation and discussion of these observations integrate the current knowledge of the structure and function of (1-->3)-beta-D-glucans.

  • 26. Phillips, R S
    et al.
    Chen, H Y
    Shim, D
    Lima, S
    Tavakoli, Khadijeh
    KTH, Tidigare Institutioner, Bioteknologi.
    Sundararaju, B
    Role of Lysine-256 in Citrobacter freundii tyrosine phenol-lyase in monovalent cation activation2004Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, nr 45, s. 14412-14419Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Tyrosine phenol-lyase (TPL) from Citrobacter freundii is dependent on monovalent cations, K+ or NH4+, for high activity. We have shown previously that Glu-69, which is a ligand to the bound cation, is important in monovalent cation binding and activation [Sundararaju, B., Chen, H., Shillcutt, S., and Phillips, R. S. (2000) Biochemistry 39, 8546-8555]. Lys-256 is located in the monovalent cation binding site of TPL, where it forms a hydrogen bond with a structural water bound to the cation. This lysine residue is highly conserved in sequences of TPL and the paralogue, tryptophan indole-lyase. We have now prepared K256A, K256H, K256R, and E69D/K256R mutant TPLs to probe the role of Lys-256 in monovalent cation binding and activation. K256A and K256H TPLs have low activity (k(cat)/K-m values of 0.01-0.1%), are not activated by monovalent cations, and do not exhibit fluorescence emission at 500 nm from the PLP cofactor. In contrast, K256R TPL has higher activity (k(cat)/K-m about 10% of wild-type TPL), is activated by K+, and exhibits fluorescence emission from the PLP cofactor. K256A, K256H, and K256R TPLs bind PLP somewhat weaker than wild-type TPL. E69D/K256R TPL was prepared to determine if the guanidine side chain could substitute for the monovalent cation. This mutant TPL has wild-type activity with S-Et-L-CYS or S-(o-nitrophenyl)-L-Cys but has no detectable activity with L-Tyr. E69D/K256R TPL is not activated by monovalent cations and does not show PLP fluorescence. In contrast to wild-type and other mutant TPLs, PLP binding to E69D/K256R is very slow, requiring several hours of incubation to obtain I mol of PLP per subunit. Thus, E69D/K256R TPL appears to have altered dynamics. All of the mutant TPLs react with inhibitors, L-Ala, L-Met, and L-Phe, to form equilibrating mixtures of external aldimine and quinonoid intermediates. Thus, Lys-256 is not the base which removes the alpha-proton during catalysis. The results show that the function of Lys-256 in TPL is in monovalent cation binding and activation.

  • 27.
    Rudsander, Ulla
    et al.
    KTH, Skolan för bioteknologi (BIO).
    Sandström, Corine
    Piens, Kathleen
    Master, Emma
    Wilson, David
    Brumer, Harry
    Kenne, Lennart
    Teeri, Tuula
    Comparative NMR analysis of cello-oligosaccharide hydrolysis by family GH9 endoglucanases from Thermobifida fusca and Populus tremula x tremuloides MichInngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995Artikkel i tidsskrift (Fagfellevurdert)
  • 28. Schagerlöf, Ulrika
    et al.
    Elmlund, Hans
    KTH, Skolan för teknik och hälsa (STH), Strukturell bioteknik.
    Gakh, Oleksandr
    Nordlund, Gustav
    Hebert, Hans
    KTH, Skolan för teknik och hälsa (STH), Strukturell bioteknik.
    Lindahl, Martin
    KTH, Skolan för teknik och hälsa (STH), Strukturell bioteknik.
    Isaya, Grazia
    Al-Karadaghi, Salam
    Structural basis of the iron storage function of frataxin from single-particle reconstruction of the iron-loaded oligomer2008Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, nr 17, s. 4948-4954Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The mitochondrial protein frataxin plays a central role in mitochondrial iron homeostasis, and frataxin deficiency is responsible for Friedreich ataxia, a neurodegenerative and cardiac disease that affects 1 in 40000 children. Here we present a single-particle reconstruction from cryoelectron microscopic images of iron-loaded 24-subunit oligomeric frataxin particles at 13 and 17 angstrom resolution. Computer-aided classification of particle images showed heterogeneity in particle size, which was hypothesized to result from gradual accumulation of iron within the core structure. Thus, two reconstructions were created from two classes of particles with iron cores of different sizes. The reconstructions show the iron core of frataxin for the first time. Compared to the previous reconstruction of iron-free particles from negatively stained images, the higher resolution of the present reconstruction allowed a more reliable analysis of the overall three-dimensional structure of the 24-meric assembly. This was done after docking the X-ray structure of the frataxin trimer into the EM reconstruction. The structure revealed a close proximity of the suggested ferroxidation sites of different monomers to the site proposed to serve in iron nucleation and mineralization. The model also assigns a new role to the N-terminal helix of frataxin in controlling the channel at the 4-fold axis of the 24-subunit oligomer. The reconstructions show that, together with some common features, frataxin has several unique features which distinguish it from ferritin. These include the overall organization of the oligomers, the way they are stabilized, and the mechanisms of iron core nucleation.

  • 29.
    Seashore-Ludlow, Brinton
    et al.
    Karolinska Inst, Sci Life Labs, Chem Biol Consortium Sweden, SE-17165 Solna, Sweden.;Karolinska Inst, Dept Med Biochem & Biophys, SE-17165 Solna, Sweden..
    Axelsson, Hanna
    Karolinska Inst, Sci Life Labs, Chem Biol Consortium Sweden, SE-17165 Solna, Sweden.;Karolinska Inst, Dept Med Biochem & Biophys, SE-17165 Solna, Sweden..
    Almqvist, Helena
    Karolinska Inst, Sci Life Labs, Chem Biol Consortium Sweden, SE-17165 Solna, Sweden.;Karolinska Inst, Dept Med Biochem & Biophys, SE-17165 Solna, Sweden..
    Dahlgren, Björn
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi.
    Jonsson, Mats
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Kemi.
    Lundback, Thomas
    Karolinska Inst, Sci Life Labs, Chem Biol Consortium Sweden, SE-17165 Solna, Sweden.;Karolinska Inst, Dept Med Biochem & Biophys, SE-17165 Solna, Sweden.;AstraZeneca, IMED Biotech Unit, Discovery Sci, Mechanist Biol & Profiling, SE-43183 Molndal, Sweden..
    Quantitative Interpretation of Intracellular Drug Binding and Kinetics Using the Cellular Thermal Shift Assay2018Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 57, nr 48, s. 6715-6725Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Evidence of physical interaction with the target protein is essential in the development of chemical probes and drugs. The cellular thermal shift assay (CETSA) allows evaluation of drug binding in live cells but lacks a framework to support quantitative interpretations and comparisons with functional data. We outline an experimental platform for such analysis using human kinase p38 alpha. Systematic variations to the assay's characteristic heat challenge demonstrate an apparent loss of compound potency with an increase in duration or temperature, in line with expectations from the literature for thermal shift assays. Importantly, data for five structurally diverse inhibitors can be quantitatively explained using a simple model of linked equilibria and published binding parameters. The platform further distinguishes between ligand mechanisms and allows for quantitative comparisons of drug binding affinities and kinetics in live cells and lysates. We believe this work has broad implications in the appropriate use of the CETSA for target and compound validation.

  • 30.
    Sevastik, Robin
    et al.
    Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University.
    Whitman, Christian P.
    Division of Medicinal Chemistry, College of Pharmacy, University of Texas.
    Himo, Fahmi
    Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University.
    Reaction Mechanism of cis-3-Chloroacrylic Acid Dehalogenase: A Theoretical Study2009Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, nr 40, s. 9641-9649Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The reaction mechanism of cis-3-chloroacrylic acid dehalogenase (cis-CaaD) is studied using the B3LYP density functional theory method. This enzyme catalyzes the hydrolytic dehalogenation of cis-3-chloroacrylic acid to yield malonate semialdehyde and HCl. The uncatalyzed reaction is first considered, and excellent agreement is found between the calculated barrier and the measured rate constant. The enzymatic reaction is then studied with an active site model consisting of 159 atoms. The results suggest an alternative mechanism for cis-CaaD catalysis and different roles for some active site residues in this mechanism.

  • 31. Spinka, M.
    et al.
    Seiferheld, S.
    Zimmermann, P.
    Bergner, E.
    Blume, A. -K
    Schierhorn, A.
    Reichenbach, Tom
    KTH, Skolan för bioteknologi (BIO), Industriell bioteknologi.
    Pertermann, R.
    Ehrt, C.
    König, S.
    Significance of Individual Residues at the Regulatory Site of Yeast Pyruvate Decarboxylase for Allosteric Substrate Activation2017Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 56, nr 9, s. 1285-1298Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The catalytic activity of the allosteric enzyme pyruvate decarboxylase from yeast is strictly controlled by its own substrate pyruvate via covalent binding at a separate regulatory site. Kinetic studies, chemical modifications, cross-linking, small-angle X-ray scattering, and crystal structure analyses have led to a detailed understanding of the substrate activation mechanism at an atomic level with C221 as the core moiety of the regulatory site. To characterize the individual role of the residues adjacent to C221, we generated variants H92F, H225F, H310F, A287G, S311A, and C221A/C222A. The integrity of the protein structure of the variants was established by small-angle X-ray scattering measurements. The analyses of both steady state and transient kinetic data allowed the identification of the individual roles of the exchanged side chains during allosteric enzyme activation. In each case, the kinetic pattern of activation was modulated but not completely abolished. Despite the crucial role of C221, the covalent binding of pyruvate is not obligate for enzyme activation but is a requirement for a kinetically efficient transition from the inactive to the active state. Moreover, only one of the three histidines guiding the activator molecule to the binding pocket, H310, specifically interacts with C221. H310 stabilizes the thiolate form of C221, ensuring a rapid nucleophilic attack of the thiolate sulfur on C2 of the regulatory pyruvate, thus forming a regulatory dyad. The influence of the other two histidines is less pronounced. Substrate activation is slightly weakened for A287G and significantly retarded for S311A.

  • 32. Wohlfahrt, G.
    et al.
    Pellikka, T.
    Boer, H.
    Teeri, Tuula T.
    KTH, Tidigare Institutioner                               , Bioteknologi.
    Koivula, A.
    Probing pH-dependent functional elements in proteins: Modification of carboxylic acid pairs in Trichoderma reesei cellobiohydrolase Cel6A2003Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 42, nr 34, s. 10095-10103Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Two carboxylic acid side chains can, depending on their geometry and environment, share a proton in a hydrogen bond and form a carboxyl-carboxylate pair. In the Trichoderma reesei cellobiohydrolase Cel6A structure, five carboxyl-carboxylate pairs are observed. One of these pairs (D175-D221) is involved in catalysis, and three other pairs are found in, or close to the two surface loops covering the active site tunnel of the catalytic domain. To stabilize Cel6A at alkaline pH values, where deprotonation of the carboxylic acids leads to repulsion of their side chains, we designed two mutant enzymes. In the first mutant, one carboxyl-carboxylate pair (E107-E399) was replaced by a corresponding amide-carboxylate pair (Q107-E399), and in the second mutant, all three carboxyl-carboxylate pairs (E107-E399, D170-E184, and D366-D419) were mutated in a similar manner. The unfolding studies using both intrinsic tryptophan fluorescence and far-ultraviolet circular dichroism spectroscopy at different pH values demonstrate that the unfolding temperature (T-m) of both mutants has changed, resulting in destabilization of the mutant enzymes at acidic pH and stabilization at alkaline pH. The effect of stabilization seems additive, as a Cel6A triple mutant is the most stable enzyme variant. This increased stability is also reflected in the 2- or 4-fold increased half-life of the two mutants at alkaline pH, while the catalytic rate on cellotetraose (at t = 0) has not changed. Increased operational stability at alkaline pH was also observed on insoluble cellulosic substrates. Local conformational changes are suggested to take place in the active site loops of Cel6A wild-type enzyme at elevated pHs (pH 7), affecting to the end-product spectrum on insoluble cellulose. The triple mutant does not show such pH-dependent behavior. Overall, our results demonstrate that carboxyl-carboxylate pair engineering is a useful tool to alter pH-dependent protein behavior.

  • 33. Wulff, Ragna Peterson
    et al.
    Lundqvist, Joakim
    Rutsdottir, Gudrun
    Hansson, Andreas
    Stenbaek, Anne
    Elmlund, Dominika
    KTH, Skolan för teknik och hälsa (STH), Strukturell bioteknik.
    Elmlund, Hans
    Jensen, Poul Erik
    Hansson, Mats
    The Activity of Barley NADPH-Dependent Thioredoxin Reductase C Is Independent of the Oligomeric State of the Protein: Tetrameric Structure Determined by Cryo-Electron Microscopy2011Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 50, nr 18, s. 3713-3723Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Thioredoxin and thioredoxin reductase can regulate cell metabolism through redox regulation of disulfide bridges or through removal of H2O2. These two enzymatic functions are combined in NADPH-dependent thioredoxin reductase C (NTRC), which contains an N-terminal thioredoxin reductase domain fused with a C-terminal thioredoxin domain. Rice NTRC exists in different oligomeric states, depending on the absence or presence of its NADPH cofactor. It has been suggested that the different oligomeric states may have diverse activity. Thus, the redox status of the chloroplast could influence the oligomeric state of NTRC and thereby its activity. We have characterized the oligomeric states of NTRC from barley (Hard rum vulgare L.). This also includes a structural model of the tetrameric NTRC derived from cryo-electron microscopy and single-particle reconstruction. We conclude that the tetrameric NTRC is a dimeric arrangement of two NTRC homodimers. Unlike that of rice NTRC, the quaternary structure of barley NTRC complexes is unaffected by addition of NADPH. The activity of NTRC was tested with two different enzyme assays. The N-terminal part of NTRC was tested in a thioredoxin reductase assay. A peroxide sensitive Mg-protoporphyrin IX monomethyl ester (MPE) cyclase enzyme system of the chlorophyll biosynthetic pathway was used to test the catalytic ability of both the N- and C-terminal parts of NTRC. The different oligomeric assembly states do not exhibit significantly different activities. Thus, it appears that the activities are independent of the oligomeric state of barley NTRC.

  • 34.
    Zou, Rongfeng
    et al.
    East China Univ Sci & Technol, Sch Chem & Mol Engn, Key Lab Adv Mat, Shanghai 200237, Peoples R China.;East China Univ Sci & Technol, Sch Chem & Mol Engn, Inst Fine Chem, Shanghai 200237, Peoples R China.;KTH Royal Inst Technol, Div Theoret Chem & Biol, Sch Biotechnol, SE-10691 Stockholm, Sweden..
    Zhu, Xiaomin
    East China Univ Sci & Technol, Sch Chem & Mol Engn, Key Lab Adv Mat, Shanghai 200237, Peoples R China.;East China Univ Sci & Technol, Sch Chem & Mol Engn, Inst Fine Chem, Shanghai 200237, Peoples R China..
    Tu, Yaoquan
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Teoretisk kemi och biologi.
    Wu, Junchen
    East China Univ Sci & Technol, Sch Chem & Mol Engn, Key Lab Adv Mat, Shanghai 200237, Peoples R China.;East China Univ Sci & Technol, Sch Chem & Mol Engn, Inst Fine Chem, Shanghai 200237, Peoples R China.;Univ Calif Berkeley, Dept Chem & Biomol Engn, 476 Stanley Hall, Berkeley, CA 94720 USA..
    Landry, Markita P.
    Univ Calif Berkeley, Dept Chem & Biomol Engn, 476 Stanley Hall, Berkeley, CA 94720 USA.;Chan Zuckerberg Biohub, San Francisco, CA USA.;Univ Calif Berkeley, Calif Inst Quantitat Biosci Qb3, Berkeley, CA 94720 USA..
    Activity of Antimicrobial Peptide Aggregates Decreases with Increased Cell Membrane Embedding Free Energy Cost2018Inngår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 57, nr 18, s. 2606-2610Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Antimicrobial peptides (AMPs) are a promising alternative to antibiotics for mitigating bacterial infections, in light of increasing bacterial resistance to antibiotics. However, predicting, understanding, and controlling the antibacterial activity of AMPs remain a significant challenge. While peptide intramolecular interactions are known to modulate AMP antimicrobial activity, peptide intermolecular interactions remain elusive in their impact on peptide bioactivity. Herein, we test the relationship between AMP intermolecular interactions and antibacterial efficacy by controlling AMP intermolecular hydrophobic and hydrogen bonding interactions. Molecular dynamics simulations and Gibbs free energy calculations in concert with experimental assays show that increasing intermolecular interactions via interpeptide aggregation increases the energy cost for the peptide to embed into the bacterial cell membrane, which in turn decreases the AMP antibacterial activity. Our findings provide a route for predicting and controlling the antibacterial activity of AMPs against Gram-negative bacteria via reductions of intermolecular AMP interactions.

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