Mechanistic and Structural Insight to an Evolved Benzoylformate Decarboxylase with Enhanced Pyruvate Decarboxylase Activity
Autor: | Michael J. McLeish, Cindy Wechsler, Kai Tittmann, Megan P. Rogers, Forest H. Andrews, Danilo Meyer |
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Jazyk: | angličtina |
Rok vydání: | 2016 |
Předmět: |
0301 basic medicine
enzyme evolution Stereochemistry Decarboxylation Context (language use) lcsh:Chemical technology Catalysis Benzoylformate decarboxylase Pyruvate decarboxylase activity lcsh:Chemistry 03 medical and health sciences NMR spectroscopy Thiamin diphosphate lcsh:TP1-1185 Enzyme kinetics Physical and Theoretical Chemistry Saturated mutagenesis X-ray crystallography 030102 biochemistry & molecular biology Chemistry Substrate (chemistry) 030104 developmental biology Biochemistry lcsh:QD1-999 Pyruvate decarboxylase |
Zdroj: | Catalysts, Vol 6, Iss 12, p 190 (2016) Catalysts; Volume 6; Issue 12; Pages: 190 |
ISSN: | 2073-4344 |
Popis: | Benzoylformate decarboxylase (BFDC) and pyruvate decarboxylase (PDC) are thiamin diphosphate-dependent enzymes that share some structural and mechanistic similarities. Both enzymes catalyze the nonoxidative decarboxylation of 2-keto acids, yet differ considerably in their substrate specificity. In particular, the BFDC from P. putida exhibits very limited activity with pyruvate, whereas the PDCs from S. cerevisiae or from Z. mobilis show virtually no activity with benzoylformate (phenylglyoxylate). Previously, saturation mutagenesis was used to generate the BFDC T377L/A460Y variant, which exhibited a greater than 10,000-fold increase in pyruvate/benzoylformate substrate utilization ratio compared to that of wtBFDC. Much of this change could be attributed to an improvement in the Km value for pyruvate and, concomitantly, a decrease in the kcat value for benzoylformate. However, the steady-state data did not provide any details about changes in individual catalytic steps. To gain insight into the changes in conversion rates of pyruvate and benzoylformate to acetaldehyde and benzaldehyde, respectively, by the BFDC T377L/A460Y variant, reaction intermediates of both substrates were analyzed by NMR and microscopic rate constants for the elementary catalytic steps were calculated. Herein we also report the high resolution X-ray structure of the BFDC T377L/A460Y variant, which provides context for the observed changes in substrate specificity. peerReviewed |
Databáze: | OpenAIRE |
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