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X-Ray structure of Candida antarctica lipase A shows a novel lid structure and a likely mode of interfacial activation
1Department of Cell and Molecular Biology, Uppsala University, Biomedical Center.
Stockholm University, Faculty of Science, Department of Organic Chemistry.
1Department of Cell and Molecular Biology, Uppsala University, Biomedical Center.
1Department of Cell and Molecular Biology, Uppsala University, Biomedical Center.
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2008 (English)In: Journal of Molecular Biology, ISSN 0022-2836, Vol. 376, no 1, 109-119 p.Article in journal (Refereed) Published
Abstract [en]

In nature, lipases (EC 3.1.1.3) catalyze the hydrolysis of triglycerides to form glycerol and fatty acids. Under the appropriate conditions, the reaction is reversible, and so biotechnological applications commonly make use of their capacity for esterification as well as for hydrolysis of a wide variety of compounds. In the present paper, we report the X-ray structure of lipase A from Candida antarctica, solved by single isomorphous replacement with anomalous scattering, and refined to 2.2-Å resolution. The structure is the first from a novel family of lipases. Contrary to previous predictions, the fold includes a well-defined lid as well as a classic α/β hydrolase domain. The catalytic triad is identified as Ser184, Asp334 and His366, which follow the sequential order considered to be characteristic of lipases; the serine lies within a typical nucleophilic elbow. Computer docking studies, as well as comparisons to related structures, place the carboxylate group of a fatty acid product near the serine nucleophile, with the long lipid tail closely following the path through the lid that is marked by a fortuitously bound molecule of polyethylene glycol. For an ester substrate to bind in an equivalent fashion, loop movements near Phe431 will be required, suggesting the primary focus of the conformational changes required for interfacial activation. Such movements will provide virtually unlimited access to solvent for the alcohol moiety of an ester substrate. The structure thus provides a basis for understanding the enzyme's preference for acyl moieties with long, straight tails, and for its highly promiscuous acceptance of widely different alcohol and amine moieties. An unconventional oxyanion hole is observed in the present structure, although the situation may change during interfacial activation

Place, publisher, year, edition, pages
Elsevier , 2008. Vol. 376, no 1, 109-119 p.
Keyword [en]
lipase; interfacial activation; hydrolase; X-ray structure; substrate specificity
National Category
Organic Chemistry
Identifiers
URN: urn:nbn:se:su:diva-13671DOI: doi:10.1016/j.jbm.2007.10.079ISI: 000253181500011OAI: oai:DiVA.org:su-13671DiVA: diva2:180191
Available from: 2008-12-03 Created: 2008-12-03 Last updated: 2010-12-15Bibliographically approved
In thesis
1. Protein Engineering of Candida antarctica Lipase A: Enhancing Enzyme Properties by Evolutionary and Semi-Rational Methods
Open this publication in new window or tab >>Protein Engineering of Candida antarctica Lipase A: Enhancing Enzyme Properties by Evolutionary and Semi-Rational Methods
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Enzymes are gaining increasing importance as catalysts for selective transformations in organic synthetic chemistry. The engineering and design of enzymes is a developing, growing research field that is employed in biocatalysis. In the present thesis, combinatorial protein engineering methods are applied for the development of Candida antarctica lipase A (CALA) variants with broader substrate scope and increased enantioselectivity. Initially, the structure of CALA was deduced by manual modelling and later the structure was established by X-ray crystallography. The elucidation of the structure of CALA revealed several biocatalytically interesting features. With the knowledge derived from the enzyme structure, enzyme variants were produced via iterative saturation mutagenesis (ISM), a powerful protein engineering approach. Several of these variants were highly active and enantioselective towards bulky esters. Furthermore, an extensively combinatorial protein engineering approach was developed and investigated. A CALA variant with a spacious substrate binding pocket that can accommodate an unusually bulky substrate, an ester derivate of the non-steroidal anti-inflammatory drug (S)-ibuprofen, was obtained with this approach.

Place, publisher, year, edition, pages
Stockholm: Department of Organic Chemistry, Stockholm University, 2010. 70 p.
Keyword
lipase, protein engineering, directed evolution, kinetic resolution, structural biology
National Category
Biocatalysis and Enzyme Technology
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:su:diva-49248 (URN)978-91-7447-202-8 (ISBN)
Public defence
2011-01-28, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
At the time of the doctoral defence the following paper was unpublished and had a status as follows: Paper nr. 5: ManuscriptAvailable from: 2011-01-03 Created: 2010-12-13 Last updated: 2011-02-21Bibliographically approved

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