Active-Site Engineering of Benzaldehyde Lyase Shows That a Point Mutation Can Confer Both New Reactivity and Susceptibility to Mechanism-Based Inhibition

Benzaldehyde lyase (BAL) from Pseudomonas putida is a thiamin diphosphate (ThDP)-dependent enzyme that catalyzes the breakdown of (R)-benzoin. Here we report that a point mutant, BAL A28S, not only catalyzes the decarboxylation of benzoylformate but, like benzoylformate decarboxylase (BFDC), is also...

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Bibliographic Details
Published in:Journal of the American Chemical Society 2010-01, Vol.132 (2), p.438-439
Main Authors: Brandt, Gabriel S, Kneen, Malea M, Petsko, Gregory A, Ringe, Dagmar, McLeish, Michael J
Format: Article
Language:English
Subjects:
Aldehyde-Lyases - antagonists & inhibitors
Aldehyde-Lyases - chemistry
Aldehyde-Lyases - metabolism
BASIC BIOLOGICAL SCIENCES
BENZALDEHYDE
BREAKDOWN
CARBON DIOXIDE
Catalytic Domain
Crystallography, X-Ray
DECARBOXYLASES
DECARBOXYLATION
Enzyme Inhibitors - chemistry
Enzyme Inhibitors - pharmacology
ENZYMES
GENE MUTATIONS
GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE
INACTIVATION
LYASES
Models, Molecular
Organophosphonates - chemistry
Organophosphonates - pharmacology
PHOSPHORYLATION
Point Mutation
Protein Engineering
PSEUDOMONAS
SERINE
Structure-Activity Relationship
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Description
Summary:Benzaldehyde lyase (BAL) from Pseudomonas putida is a thiamin diphosphate (ThDP)-dependent enzyme that catalyzes the breakdown of (R)-benzoin. Here we report that a point mutant, BAL A28S, not only catalyzes the decarboxylation of benzoylformate but, like benzoylformate decarboxylase (BFDC), is also inactivated by the benzoylformate analogues methyl benzoylphosphonate (MBP) and benzoylphosphonate (BP). The latter has no effect on wild-type BAL, and the inactivation of the A28S variant is shown to result from phosphorylation of the newly introduced serine residue. This lends support to the proposal that an appropriately placed nucleophile facilitates the expulsion of carbon dioxide from the active site in many ThDP-dependent decarboxylases.
ISSN:0002-7863
1520-5126
DOI:10.1021/ja907064w