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Creating Space for Large Secondary Alcohols by Rational Redesign of Candida antarctica Lipase B
KTH, Skolan för bioteknologi (BIO).
KTH, Skolan för bioteknologi (BIO).
KTH, Skolan för bioteknologi (BIO).
KTH, Skolan för bioteknologi (BIO).
2005 (engelsk)Inngår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, nr 6, s. 1051-1056Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

The active site of Candida antarctica lipase B (CALB) hosts the catalytic triad (Ser-His-Asp), an oxyanion hole and a stereospecificity pocket. During catalysis, the fast-reacting enantiomer of secondary alcohols places its medium-sized substituent in the stereospecificity pocket and its large substituent towards the active-site entrance. The largest group to fit comfortably in the stereospecificity pocket is ethyl, and this restricts the number of secondary alcohols that are good substrates for CALB. In order to overcome this limitation, the size of the stereospecificity pocket was redesigned by changing Trp104. The substrate specificity of the Trp104Ala mutant compared to that of the wild-type lipase increased 270 times towards heptan-4-ol and 5500 times towards nonan-5-ol; this resulted in the high specificity constants 1100 and 830 s(-1)m(-1), respectively. The substrate selectivity changed over 400000 times for nonan-5-ol over propan-2-ol with both Trp104Ala and the Trp104Gln mutations.

sted, utgiver, år, opplag, sider
2005. Vol. 6, nr 6, s. 1051-1056
Emneord [en]
enzyme catalysis, protein engineering, rational design, specificity constant, substrate specificity
HSV kategori
Identifikatorer
URN: urn:nbn:se:kth:diva-5091DOI: 10.1002/cbic.200400410ISI: 000229730000016PubMedID: 15883973Scopus ID: 2-s2.0-20444448921OAI: oai:DiVA.org:kth-5091DiVA, id: diva2:7848
Merknad
QC 20100907. Uppdaterad från In press till Published (20100907)Tilgjengelig fra: 2005-05-10 Laget: 2005-05-10 Sist oppdatert: 2020-03-09bibliografisk kontrollert
Inngår i avhandling
1. Rational redesign of Candida antarctica lipase B
Åpne denne publikasjonen i ny fane eller vindu >>Rational redesign of Candida antarctica lipase B
2005 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

This thesis describes the use of rational redesign to modify the properties of the enzyme Candida antarctica lipase B. Through carefully selected single-point mutations, we were able to introduce substrate-assisted catalysis and to alter the reaction specificity. Other single-point mutations afforded variants with greatly changed substrate selectivity and enantioselectivity.

Mutation of the catalytic serine changed the hydrolase activity into an aldolase activity. The mutation decreased the activation energy for aldol addition by 4 kJ×mol-1, while the activation energy increased so much for hydrolysis that no hydrolysis activity could be detected. This mutant can catalyze aldol additions that no natural aldolases can catalyze.

Mutation of the threonine in the oxyanion hole proved the great importance of its hydroxyl group in the transition-state stabilization. The lost transition-state stabilization was partly replaced through substrate-assisted catalysis with substrates carrying a hydroxyl group. The poor selectivity of the wild-type lipase for ethyl 2-hydroxypropanoate (E=1.6) was greatly improved in the mutant (E=22), since only one enantiomer could perform substrate-assisted catalysis.

The redesign of the size of the stereospecificity pocket was very successful. Mutation of the tryptophan at the bottom of this pocket removed steric interactions with secondary alcohols that have to position a substituent larger than an ethyl in this pocket. This mutation increased the activity 5 500 times towards 5-nonanol and 130 000 times towards (S)-1-phenylethanol. The acceptance of such large substituents (butyl and phenyl) in the redesigned stereospecificity pocket increases the utility of lipases in biocatalysis. The improved activity with (S)-1-phenylethanol strongly contributed to the 8 300 000 times change in enantioselectivity towards 1-phenylethanol; example of such a large change was not found in the literature. The S-selectivity of the mutant is unique for lipases. Its enantioselectivity increases strongly with temperature reaching a useful S-selectivity (E=44) at 69 °C.

Thermodynamics analysis of the enantioselectivity showed that the mutation in the stereospecificity pocket mainly changed the entropic term, while the enthalpic term was only slightly affected. This pinpoints the importance of entropy in enzyme catalysis and entropy should not be neglected in rational redesign.

Emneord
Biochemistry, Candida antarctica lipase B, rational redesign, secondary alcohols, substrate-assisted catalysis, S-selective, entropy, aldolase, stereospecificity pocket, oxyanion hole., Biokemi
HSV kategori
Identifikatorer
urn:nbn:se:kth:diva-186 (URN)91-7178-012-2 (ISBN)
Disputas
2005-05-13, FR4, AlbaNova, Roslagstullsbacken 21, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2005-05-10 Laget: 2005-05-10 Sist oppdatert: 2012-03-21bibliografisk kontrollert

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