Engineering Mesorhizobium loti pyridoxamine–pyruvate aminotransferase for production of pyridoxamine with l-glutamate as an amino donor

Pyridoxamine–pyruvate aminotransferase (PPAT), a novel pyridoxal 5′-phosphate-independent aminotransferase, reversibly catalyzes the transfer of an amino group between pyridoxamine and pyruvate to generate pyridoxal and l-alanine. The enzyme can be used for synthesis of pyridoxamine, a promising candidate for prophylaxis and treatment of diabetic complications. A disadvantage of PPAT for industrial application to the synthesis is that it requires an expensive amino acid l-alanine as an amino donor. Here, mutated PPATs with a high activity toward 2-oxoglutarate (and hence toward l-glutamate) were prepared by a rational design plus random mutagenesis of the wild-type PPAT because l-glutamate is readily and economically available. The PPAT(Y35H/V70R/F247C) showed 9.1-fold lower Km and 4.3-fold higher kcat values than those of the wild-type PPAT. The model of the complex of mutated PPAT and pyridoxyl-l-glutamate showed that γ-carboxyl group of l-glutamate was hydrogen-bound with an imidazole group of His35. The production of pyridoxamine from pyridoxal with transformed Escherichia coli cells expressing the mutated PPAT did not correlate with the kcat value or catalytic efficiency of the mutated PPAT but with Km value at a low level. E. coli cells expressing the PPAT(M2T/Y35H/V70K/E212G) could be used for in vitro conversion of pyridoxal into pyridoxamine at 30 °C with l-glutamate as an amino donor.

A model of the active site of a complex of PPAT(Y35H/V70R/F247C) and pyridoxyl-l-glutamate.Figure optionsDownload as PowerPoint slideResearch highlightsIn this study, the rational design and the random mutagenesis were used in combination to generate mutated pyridoxamine–pyruvate aminotransferase (PPAT) with the high activity toward 2-oxoglutarate and l-glutamate. The transformed E. coli cells expressing PPAT(M2T/Y35H/V70K/E212G) could be used for in vitro conversion of pyridoxal into pyridoxamine with l-glutamate as an amino donor.