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Molecular modelling - understanding and prediction of enzyme selectivity.
KTH, School of Biotechnology (BIO). (Biochemistry)
2009 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Molecular modelling strategies for evaluation of enzyme selectivity wereinvestigated with a focus on principles of how molecular interactionscould be evaluated to provide information about selectivity. Althoughmolecular modelling provides tools for evaluation of geometrical andenergy features of molecular systems, no general strategies for evaluationof enzyme selectivity exist. Geometrical analyses can be based uponinspection and reasoning about molecular interactions, which provide aneasily accessible way to gain information, but suffer from the risk of biasput in by the modeller. They can also be based on geometrical features ofmolecular interactions such as bond lengths and hydrogen-bond formation.Energy analyses are appealing for their modeller independenceand for the possibility to predict not only stereopreference, but also itsmagnitude.In this thesis, four examples of enantio- or regioselective serinehydrolase-catalysed reaction systems are presented together with developedmodelling protocols for explanation, prediction or enhancement ofselectivity. Geometrical as well as energy-based methodology were used,and provided an understanding of the structural basis of enzymeselectivity. In total, the protocols were successful in making qualitative explanationsand predictions of stereoselectivity, although quantitative determinationswere not achieved.

Place, publisher, year, edition, pages
Stockholm: KTH , 2009. , 27 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009:11
Keyword [en]
molecular modelling; regioselectivity; enantioselectivity; molecular dynamics; serine hydrolase; stereospecificity
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-10532ISBN: 978-91-7415-325-5 (print)OAI: oai:DiVA.org:kth-10532DiVA: diva2:218880
Presentation
(English)
Supervisors
Available from: 2009-05-26 Created: 2009-05-25 Last updated: 2010-10-12Bibliographically approved
List of papers
1. Switched enantiopreference of Humicola lipase for 2-phenoxyalkanoic acid ester homologs can be rationalized by different substrate binding modes
Open this publication in new window or tab >>Switched enantiopreference of Humicola lipase for 2-phenoxyalkanoic acid ester homologs can be rationalized by different substrate binding modes
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1999 (English)In: Tetrahedron: asymmetry, ISSN 0957-4166, E-ISSN 1362-511X, Vol. 10, no 21, 4191-4202 p.Article in journal (Refereed) Published
Abstract [en]

Humicola lanuginosa lipase was used for enantioselective hydrolyses of a series of homologous 2-phenoxyalkanoic acid ethyl esters. The enantioselectivity (E-value) of the enzyme changed from an (R)-enantiomer preference for the smallest substrate, 2-phenoxypropanoic acid ester, to an (S)-enantiomer preference for the homologous esters with longer acyl moieties. The E-values span the range from E=13 (R) to E=56 (S). A molecular modeling study identified two different substrate-binding modes for each enantiomer. We found that the enantiomers favored different modes. This discovery provided a model that offered a rational explanation for the observed switch in enantioselectivity. (C) 1999 Elsevier Science Ltd. All rights reserved.

Keyword
CANDIDA-RUGOSA LIPASE, LANUGINOSA LIPASE, QUANTITATIVE-ANALYSES, SUBTILISIN CARLSBERG, NUCLEIC-ACIDS, FORCE-FIELD, ENANTIOMERS, BIOCATALYSIS, RESOLUTION, BIOTRANSFORMATIONS
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-25181 (URN)000083948700019 ()
Note
QC 20101012 NR 20140804Available from: 2010-10-12 Created: 2010-10-12 Last updated: 2017-12-12Bibliographically approved
2. Highly enantioselective kinetic resolution of two tertiary alcohols using mutants of an esterase from Bacillus subtilis
Open this publication in new window or tab >>Highly enantioselective kinetic resolution of two tertiary alcohols using mutants of an esterase from Bacillus subtilis
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2007 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 20, no 3, 125-131 p.Article in journal (Refereed) Published
Abstract [en]

Enzyme-catalyzed kinetic resolutions of secondary alcohols are a standard procedure today and several lipases and esterases have been described to show high activity and enantioselectivity. In contrast, tertiary alcohols and their esters are accepted only by a few biocatalysts. Only lipases and esterases with a conserved GGG(A)X-motif are active, but show low activity combined with low enantioselectivity in the hydrolysis of tertiary alcohol esters. We show in this work that the problematic autohydrolysis of certain compounds can be overcome by medium and substrate engineering. Thus, 3-phenylbut-1-yn-3-yl acetate was hydrolyzed by the esterase from Bacillus subtilis (BS2, mutant Gly105Ala) with an enantioselectivity of E = 56 in the presence of 20% (v/v) DMSO compared to E = 28 without a cosolvent. Molecular modeling was used to study the interactions between BS2 and tertiary alcohol esters in their transition state in the active site of the enzyme. Guided by molecular modeling, enzyme variants with highly increased enantioselectivity were created. For example, a Glu188Asp mutant converted the trifluoromethyl analog of 3-phenylbut-1-yn-3-yl acetate with an excellent enantioselectivity (E > 100) yielding the (S)-alcohol with > 99%ee. In summary, protein engineering combined with medium and substrate engineering afforded tertiary alcohols of very high enantiomeric purity.

Keyword
tertiary alcohol, enantioselectivity, enzyme catalysis, esterase, rational protein design, des molecules conjuguees, candida-antarctica, oxyanion hole, acetate esters, nucleic-acids, force-field, lipase-b, enzyme, transesterification, stereocenters
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-16506 (URN)10.1093/protein/gzm003 (DOI)000245354800004 ()2-s2.0-34047217953 (Scopus ID)
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved
3. Enantioselectivity in Candida antarctica lipase B: A molecular dynamics study
Open this publication in new window or tab >>Enantioselectivity in Candida antarctica lipase B: A molecular dynamics study
2001 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 10, no 2, 329-338 p.Article in journal (Refereed) Published
Abstract [en]

A major problem in predicting the enantioselectivity of an enzyme toward substrate molecules is that even high selectivity toward one substrate enantiomer over the other corresponds to a very small difference in free energy. However, total free energies in enzyme-substrate systems are very large and fluctuate significantly because of general protein motion. Candida antarctica lipase B (CALB), a serine hydrolase, displays enantioselectivity toward secondary alcohols. Here, we present a modeling study where the aim has been to develop a molecular dynamics-based methodology for the prediction of enantioselectivity in CALB. The substrates modeled (seven in total) were 3-methyl-2-butanol with various aliphatic carboxylic acids and also 2-butanol, as well as 3,3-dimethyl-2-butanol with octanoic acid. The tetrahedral reaction intermediate was used as a model of the transition state. Investigative analyses were performed on ensembles of nonminimized structures and focused on the potential energies of a number of subsets within the modeled systems to determine which specific regions are important for the prediction of enantioselectivity. One category of subset was based on atoms that make up the core structural elements of the transition state. We considered that a more favorable energetic conformation of such a subset should relate to a greater likelihood for catalysis to occur, thus reflecting higher selectivity. The results of this study conveyed that the use of this type of subset was viable for the analysis of structural ensembles and yielded good predictions of enantioselectivity.

Keyword
molecular dynamics, enantioselectivity, enzyme catalysis, transition state, free energy, enzymatic enantioselectivity, kinetic resolutions, pseudomonas-cepacia, secondary alcohols, nucleic-acids, force-field, stereoselectivity, enantiomers, mechanism, proteins
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-20507 (URN)10.1110/ps.33901 (DOI)000167926000011 ()11266619 (PubMedID)
Note
QC 20100525Available from: 2010-08-10 Created: 2010-08-10 Last updated: 2017-12-12Bibliographically approved
4. The modelling and kinetic investigation of the lipase-catalysed acetylation of stereoisomeric prostaglandins
Open this publication in new window or tab >>The modelling and kinetic investigation of the lipase-catalysed acetylation of stereoisomeric prostaglandins
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2005 (English)In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 35, no 1-3, 62-69 p.Article in journal (Refereed) Published
Abstract [en]

The lipase-catalysed acetylation of the hydroxyl groups of five stereoisomeric prostaglandins of type F was investigated by means of molecular dynamics simulations and the results compared with experimental observations. An NMR spectroscopic monitoring was performed to estimate reaction velocities and the regioselectivity. A molecular modelling protocol that could qualitatively differentiate between the OH groups of prostaglandins being either accessible or unaccessible to the Candida antarctica lipase B (CALB) catalysed acetylation was developed. The protocol developed analysed the protein structure deformation, the content of essential hydrogen bonds and the function-based subset energy of tetrahedral intermediates along the molecular dynamics simulations trajectory. The tetrahedral intermediates displaying a deformation RMS value lower than 3.0 angstrom, an essential hydrogen bond content over 50% and a subset energy less than -95 kJ/mol were classified active. In total, the accessibility of 16 out of 17 different prostaglandin OH groups was correctly predicted.

Keyword
lipase-catalysed acetylation, Novozym 435, low-water media, monitoring by H-1 NMR, prostaglandin, molecular dynamics simulations, enantiomerically pure diastereomers, candida-antarctica, nucleic-acids, force-field, isoprostanes, physiology, proteins, ester
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-14929 (URN)10.1016/j.molcatb.2005.05.008 (DOI)000230671100011 ()2-s2.0-21644442447 (Scopus ID)
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2017-12-12Bibliographically approved

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