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Exploiting enzyme promiscuity for rational design
KTH, School of Biotechnology (BIO), Biochemistry.
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Enzymes are today well recognized in various industrial applications, being an important component in detergents, and catalysts in the production of agrochemicals, foods, pharmaceuticals, and fine chemicals. Their large use is mainly due to their high selectivity and environmental advantage, compared to traditional catalysts. Tools and techniques in molecular biology offer the possibility to screen the natural sources and engineer new enzyme activities which further increases their usefulness as catalysts, in a broader area.

Although enzymes show high substrate and reaction selectivity many enzymes are today known to catalyze other reactions than their natural ones. This is called enzyme promiscuity. It has been suggested that enzyme promiscuity is Nature’s way to create diversity. Small changes in the protein sequence can give the enzyme new reaction specificity.

In this thesis I will present how rational design, based on molecular modeling, can be used to explore enzyme promiscuity and to change the enzyme reaction specificity. The first part of this work describes how Candida antarctica lipase B (CALB), by a single point mutation, was mutated to give increased activity for aldol additions, Michael additions and epoxidations. The activities of these reactions were predicted by quantum chemical calculations, which suggested that a single-point mutant of CALB would catalyze these reactions. Hence, the active site of CALB, which consists of a catalytic triad (Ser, His, Asp) and an oxyanion hole, was targeted by site-directed mutagenesis and the nucleophilic serine was mutated for either glycine or alanine. Enzymes were expressed in Pichia pastoris and analyzed for activity of the different reactions. In the case of the aldol additions the best mutant showed a four-fold initial rate over the wild type enzyme, for hexanal. Also Michael additions and epoxidations were successfully catalyzed by this mutant.

In the last part of this thesis, rational design of alanine racemase from Geobacillus stearothermophilus was performed in order to alter the enzyme specificity. Active protein was expressed in Escherichia coli and analyzed. The explored reaction was the conversion of alanine to pyruvate and 2-butanone to 2-butylamine. One of the mutants showed increased activity for transamination, compared to the wild type.

Place, publisher, year, edition, pages
Stockholm: KTH , 2005. , 58 p.
Keyword [en]
Biochemistry, Candida antarctica lipase B, alanine racemase, substrate specificity, reaction specificity, enzyme catalytic promiscuity
Keyword [sv]
Biokemi
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:kth:diva-199ISBN: 91-7178-008-4 (print)OAI: oai:DiVA.org:kth-199DiVA: diva2:7882
Public defence
2005-05-20, Oskar Kleins auditorium, AlvaNova, 13:00 (English)
Opponent
Supervisors
Note
QC 20100929Available from: 2005-05-15 Created: 2005-05-15 Last updated: 2010-09-29Bibliographically approved
List of papers
1. Carbon-Carbon Bonds by Hydrolytic Enzymes
Open this publication in new window or tab >>Carbon-Carbon Bonds by Hydrolytic Enzymes
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2003 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 125, no 4, 874-875 p.Article in journal (Refereed) Published
Abstract [en]

Enzymes are efficient catalysts in synthetic chemistry, and their catalytic activity with unnatural substrates in organic reaction media is an area attracting much attention. Protein engineering has opened the possibility to change the reaction specificity of enzymes and allow for new reactions to take place in their active sites. We have used this strategy on the well-studied active-site scaffold offered by the serine hydrolase Candida antarctica lipase B (CALB, EC 3.1.1.3) to achieve catalytic activity for aldol reactions. The catalytic reaction was studied in detail by means of quantum chemical calculations in model systems. The predictions from the quantum chemical calculations were then challenged by experiments. Consequently, Ser105 in CALB was targeted by site-directed mutagenesis to create enzyme variants lacking the nucleophilic feature of the active site. The experiments clearly showed an increased reaction rate when the aldol reaction was catalyzed by the mutant enzymes as compared to the wild-type lipase. We expect that the new catalytic activity, harbored in the stable protein scaffold of the lipase, will allow aldol additions of substrates, which cannot be reached by traditional aldolases

National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-5090 (URN)10.1021/ja028056b (DOI)000180579600011 ()
Note
QC 20100901Available from: 2005-05-10 Created: 2005-05-10 Last updated: 2017-12-05Bibliographically approved
2. Aldol Additions with Mutant Lipase: Analysis by Experiments and Theoretical Calculations
Open this publication in new window or tab >>Aldol Additions with Mutant Lipase: Analysis by Experiments and Theoretical Calculations
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2004 (English)In: Journal of Molecular Catalysis B: Enzymatic, ISSN 1381-1177, E-ISSN 1873-3158, Vol. 31, no 4-6, 123-128 p.Article in journal (Refereed) Published
Abstract [en]

A Ser105Ala mutant of Candida antarctica lipase B has previously been shown to catalyze aldol additions. Quantum chemical calculations predicted a reaction rate similar to that of natural enzymes, whereas experiments showed a much lower reaction rate. Molecular dynamics simulations, presented here, show that the low reaction rate is a consequence of the low frequencies of near attack complexes in the enzyme. Equilibrium was also considered as a reason for the slow product formation, but could be excluded by performing a sequential reaction to push the reaction towards product formation. In this paper, further experimental results are also presented, highlighting the importance of the entire active site for catalysis.

Keyword
Ab initio calculations; Aldolase; Lipase; Oxyanion hole; Reaction specificity
National Category
Biological Sciences Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-5112 (URN)10.1016/j.molcatb.2004.08.005 (DOI)000225517400008 ()2-s2.0-8644236642 (Scopus ID)
Note
QC 20100827Available from: 2005-05-15 Created: 2005-05-15 Last updated: 2017-12-04Bibliographically approved
3. Exploring the Active-Site of a Rationally Redesigned Lipase for Catalysis of Michael-Type Additions
Open this publication in new window or tab >>Exploring the Active-Site of a Rationally Redesigned Lipase for Catalysis of Michael-Type Additions
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2005 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, 331-336 p.Article in journal (Refereed) Published
Abstract [en]

Michael-type additions of various thiols and alpha,beta-unsaturated carbonyl compounds were performed in organic solvent catalyzed by wild-type and a rationally redesigned mutant of Candida antarctica lipase B. The mutant locks the nucleophilic serine 105 in the active-site; this results in a changed catalytic mechanism of the enzyme. The possibility of utilizing this mutant for Michael-type additions was initially explored by quantum-chemical calculations on the reaction between acrolein and methanethiol in a model system. The model system was constructed on the basis of docking and molecular-dynamics simulations and was designed to simulate the catalytic properties of the active site. The catalytic system was explored experimentally with a range of different substrates. The k(cat) values were found to be in the range of 10(-3) to 4 min(-1), similar to the values obtained with aldolase antibodies. The enzyme proficiency was 10(7). Furthermore, the Michael-type reactions followed saturation kinetics and were confirmed to take place in the enzyme active site.

Keyword
DENSITY-FUNCTIONAL THEORY; ENZYMATIC-REACTIONS; HYDROLYTIC ENZYMES; ALKALINE PROTEASE; BACILLUS-SUBTILIS; ORGANIC MEDIA; GROUND-STATE; MECHANISM
Identifiers
urn:nbn:se:kth:diva-5113 (URN)10.1002/cbic.200400213 (DOI)000226957100014 ()2-s2.0-20544452621 (Scopus ID)
Note
QC20100609Available from: 2005-05-15 Created: 2005-05-15 Last updated: 2017-12-04Bibliographically approved
4. Investigation of Substrate Specificity of Geobacillus stearothermophilus Alanine Racemase
Open this publication in new window or tab >>Investigation of Substrate Specificity of Geobacillus stearothermophilus Alanine Racemase
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-5114 (URN)
Note
QC 20100928Available from: 2005-05-15 Created: 2005-05-15 Last updated: 2010-09-29Bibliographically approved
5. Epoxidation catalyzed by a CALB mutant
Open this publication in new window or tab >>Epoxidation catalyzed by a CALB mutant
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-24884 (URN)
Note
QC 20100929Available from: 2010-09-29 Created: 2010-09-29 Last updated: 2010-09-29Bibliographically approved

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