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Catalysts 2016, 6(12), 190; doi:10.3390/catal6120190

Mechanistic and Structural Insight to an Evolved Benzoylformate Decarboxylase with Enhanced Pyruvate Decarboxylase Activity

1
Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202, USA
2
Göttingen Center for Molecular Biosciences, Georg-August-University Göttingen, Ernst-Caspari-Haus, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany
*
Author to whom correspondence should be addressed.
Academic Editors: Jose M. Palomo and Cesar Mateo
Received: 1 November 2016 / Revised: 25 November 2016 / Accepted: 28 November 2016 / Published: 30 November 2016
(This article belongs to the Special Issue Asymmetric and Selective Biocatalysis)
View Full-Text   |   Download PDF [2448 KB, uploaded 30 November 2016]   |  

Abstract

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. View Full-Text
Keywords: Thiamin diphosphate; X-ray crystallography; NMR spectroscopy; enzyme evolution Thiamin diphosphate; X-ray crystallography; NMR spectroscopy; enzyme evolution
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Andrews, F.H.; Wechsler, C.; Rogers, M.P.; Meyer, D.; Tittmann, K.; McLeish, M.J. Mechanistic and Structural Insight to an Evolved Benzoylformate Decarboxylase with Enhanced Pyruvate Decarboxylase Activity. Catalysts 2016, 6, 190.

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