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

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 K m value for pyruvate and, concomitantly, a decrease in the k cat 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.

Kinetics of Whole Cells and Ethanol Production from Candida tropicalis TISTR 5306 Cultivation in Batch and Fed-batch Modes Using Assorted Grade Fresh Longan Juice

“…The cytoplasmic pyruvate decarboxylase (PDC) is one of the key enzymes of yeast fermentative metabolism, such as Bacillus subtilis Candida tropicalis, C. utilis, Kluyveromyces marxianus, Pichia pastoris, Saccharomyces cerevisiae, and Aspergillus niger (Andrews et al 2016, Tangtua et al 2013, Berłowska et al 2009. PDC was the first active enzyme of the glycolytic pathway in many fermentative microorganisms and its role was generally recognized for ethanol production (Zhao et al 2015, Ward & Singh 2000.…”