Abstract
L-proline amide hydrolase (PAH, EC 3.5.1.101) is a barely described enzyme belonging to the peptidase S33 family, and is highly similar to prolyl aminopeptidases (PAP, EC. 3.4.11.5). Besides being an S-stereoselective character towards piperidine-based carboxamides, this enzyme also hydrolyses different L-amino acid amides, turning it into a potential biocatalyst within the Amidase Process. In this work, we report the characterization of L-proline amide hydrolase from Pseudomonas syringae (PsyPAH) together with the first X-ray structure for this class of L-amino acid amidases. Recombinant PsyPAH showed optimal conditions at pH 7.0 and 35 °C, with an apparent thermal melting temperature of 46 °C. The enzyme behaved as a monomer at the optimal pH. The L-enantioselective hydrolytic activity towards different canonical and non-canonical amino-acid amides was confirmed. Structural analysis suggests key residues in the enzymatic activity.
Highlights
L-proline amide hydrolase (PAH, EC 3.5.1.101) is a barely described enzyme, which up to now, has only been characterized with some detail in Pseudomonas azotoformans IAM1603 (LaaAPa ) [1,2]
PAH belongs to the serine peptidase S33 family, together with prolyl aminopeptidases (PAP, EC. 3.4.11.5) or prolinases (Pro-Xaa dipeptidase, 3.4.13.18)
An estimated Rh of 2.5 ± 0.40 nm was obtained for PsyPAH by DLS (20 mM phosphate pH 7.0)
Summary
L-proline amide hydrolase (PAH, EC 3.5.1.101) is a barely described enzyme, which up to now, has only been characterized with some detail in Pseudomonas azotoformans IAM1603 (LaaAPa ) [1,2]. PAH was suggested as a different member of this family since LaaAPa proved a different substrate scope than PAPs [2]. The enzyme proved enantioselective towards different piperidine-based carboxamides, L-prolinamide, and other different amino acid amides (Figure 1A). Process” for the industrial production of optically pure amino acids, its different substrate scope prompted its nomenclature as L-amino acid amidase [2,3,4]. This biotechnological process consists of the dynamic kinetic resolution of amino acid amides mixtures using an α-amino-ε-caprolactam racemase together with a stereoselective “D- or L-amidase”
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