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

Forest Andrews,Kai Tittmann,Michael Mcleish,Cindy Wechsler,Danilo Meyer,Megan Rogers

doi:10.3390/catal6120190

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.

Highlights

The decarboxylases form the largest group of thiamin diphosphate (ThDP)-dependent enzymes [1].The archetypal member of this group is pyruvate decarboxylase (PDC), which catalyzes the nonoxidative decarboxylation of pyruvate to yield acetaldehyde and carbon dioxide, a reaction critical to the fermentation pathway of several yeast and bacteria [2]
Our earlier study compared the reactions of wtBFDC, ScPDC and the T377L/A460Y variant only with benzoylformate and pyruvate
The Benzoylformate decarboxylase (BFDC) variant T377L/A460Y effectively acts as a “pseudo” PDC

Summary

Introduction

The decarboxylases form the largest group of thiamin diphosphate (ThDP)-dependent enzymes [1].The archetypal member of this group is pyruvate decarboxylase (PDC), which catalyzes the nonoxidative decarboxylation of pyruvate to yield acetaldehyde and carbon dioxide, a reaction critical to the fermentation pathway of several yeast and bacteria [2]. X-ray structures of PDCs from a variety of species show that, in addition to ThDP, the active site contains two ionizable acidic residues as well as two contiguous histidine residues that are located on an ordered loop [3,4,5,6]. The latter has been termed the HH-motif, and mutagenesis and kinetic studies have revealed that both histidines play significant roles in catalysis [7,8,9,10]. X-ray structures of several other ThDP-dependent decarboxylases reveal that, even though a variety of residues may line the substrate-binding pocket, thereby explaining the observed differences in substrate specificity, most possess the HH-motif and the two acidic residues [11,12,13].

Methods

Results

Conclusion