Functional characterization of PLP fold type IV transaminase with a mixed type of activity from Haliangium ochraceum

摘要

Pyridoxal-5'-phosphate (PLP)-dependent transaminases are industrially important enzymes catalyzing the stereoselective amination of ketones and keto acids. Transaminases of PLP fold type IV are characterized by (R)- or (S)-stereoselective transfer of amino groups, depending on the substrate profile of the enzyme. PLP fold type IV transaminases include branched-chain amino acid transaminases (BCATs), D-amino acid transaminases and (R)-amine:pyruvate transaminases. Recently, transaminases with a mixed type of activity were identified and characterized. Here, we report biochemical and structural characterization of a transaminase from myxobacterium Haliangium ochraceum (Hoch3033), which is active towards keto analogs of branched-chain amino acids (specific substrates for BCATs) and (R)-(+)-alpha-methylbenzylamine (specific substrate for (R)-amine:pyruvate transaminases). The enzyme is characterized by an alkaline pH optimum (pH 10.0-10.5) and a tolerance to high salt concentrations (up to 2 M NaCl). The structure of Hoch3033 was determined at 2.35 angstrom resolution. The overall fold of the enzyme was similar to those of known enzymes of PLP fold type IV. The mixed type of activity of Hoch3033 was implemented within the BCAT-like active site. However, in the active site of Hoch3033, we observed substitutions of specificity-determining residues that are important for substrate binding in canonical BCATs. We suggest that these changes result in the loss of activity towards a-ketoglutarate and increase the affinity towards (R)-(+)-alpha-methylbenzylamine. These results complement our knowledge of the catalytic diversity of transaminases and indicate the need for further research to understand the structural basis of substrate specificity in these enzymes.