Exchanging the substrate specificities of pyruvate decarboxylase from Zymomonas mobilis and benzoylformate decarboxylase from Pseudomonas putida

Abstract

Pyruvate decarboxylase from Zymomonas mobilis (PDC) and benzoylformate decarboxylase from Pseudomonas putida (BFD) are thiamine diphosphate-dependent enzymes that decarboxylate 2-keto acids. Although they share a common homotetrameric structure they have relatively low sequence similarity and different substrate spectra. PDC prefers short aliphatic substrates whereas BFD favours aromatic 2-keto acids. These preferences are also reflected in their carboligation reactions. PDC catalyses the conversion of benzaldehyde and acetaldehyde to (R)-phenylacetylcarbinol and predominantly (S)-acetoin, whereas (R)-benzoin and mainly (S)-2-hydroxypropiophenone are the products of BFD catalysis. Comparison of the X-ray structures of both enzymes identified two residues in each that were likely to be involved in determining substrate specificity. Site-directed mutagenesis was used to interchange these residues in both BFD and PDC. The substrate range and kinetic parameters for the decarboxylation reaction were studied for each variant. The most successful variants, PDCI472A and BFDA460I, catalysed the decarboxylation of benzoylformate and pyruvate, respectively, although both variants now preferred the long-chain aliphatic substrates, 2-ketopentanoic and 2-ketohexanoic acid. With respect to the carboligase activity, PDCI472A proved to be a real chimera between PDC and BFD whereas BFDA460I/F464I provided the most interesting result with an almost complete reversal of the stereochemistry of its 2-hydroxypropiophenone product.

Engineering pyruvate decarboxylase-mediated ethanol production in the thermophilic host Geobacillus thermoglucosidasius

“…The substrate recognition and decarboxylation range of the enzyme is similar to that of other Gram negative PDCs , showing a preference for short-chain aliphatic 2-keto acids (Gocke et al, 2009). The value of k cat /K M for pyruvate compared to those for 2-ketobutanoate and 2-ketopentanoate, the nearest analogues, and the retention of Ile468, thought to be involved in substrate specificity, suggests that this enzyme favors pyruvate as its physiological substrate (Siegert et al, 2005;Gocke et al, 2009). In terms of its kinetic behavior, GoxPDC appears to behave in a manner similar to other Gram-negative bacterial PDCs, displaying the same pH dependent increase in k cat /K M while catalytic efficiency (k cat ) remains largely unchanged.…”

“…Ile469 is, however, located in a region of the enzyme which may be sensitive to changes. It is positioned adjacent to Glu468, important in catalytic activity, Ile467, involved in substrate recognition, and Ile471, crucial for substrate positioning (Pohl et al, 1998;Siegert et al, 2005;Meyer et al, 2010). Figure 1).…”

Identification of Novel Benzoylformate Decarboxylases by Growth Selection

“…4.1.1.7) catalyzes the formation of benzaldehyde from benzoylformate by decarboxylation. The structure of BFD was solved in the absence (11) and the presence (34) of mandelic acid as an inhibitor, confirming that the enzyme acts as a tetramer, and active-site residues have been elucidated by site-directed mutants (34,40) and directedevolution studies (26,27).…”

“…3). (34,40), and the boxed residues are involved in thiamine diphosphate cofactor and metal binding.…”

Comparison of Purification Efficiencies of Different Affinity Separation Techniques

“…It is not fully understood, whether the carboligase activity is of physiological importance or just a not required function of the enzyme in Pseudomonas putida [3]. Wilcocks et al described the carboligase activity of BFD for the first time.…”