Serrano, D. A. | bioRxiv Science

bioRxiv2022

Structural and Preliminary Biochemical Characterization of MppQ, a PLP-Dependent Aminotransferase from Streptomyces hygroscopicus

MppQ is an enzyme of unknown function from Streptomyces hygroscopicus that is involved in the biosynthesis of the nonproteinogenic amino acid L-enduracididine (L-End). Since L-End is a component of several peptides showing high activity against methicillin-resistant Staphylococcus aureus (MRSA), a complete understanding of its biosynthetic pathway is of utmost importance for developing chemoenzymatic routes for syntheses of novel antibiotics. In this work, we report high-resolution X-ray crystal structures of MppQ complexed with pyridoxal-5-phosphate (PLP) and pyridoxamine-5-phosphate (PMP). The structure of MppQ shares a fold with known Type I PLP-dependent aminotransferases, consisting of an N-terminal extension, large domain, and a small domain. We also report the first functional characterization of MppQ, which we incubated with enzymatically produced 2-ketoenduracidine and observed conversion to L-End via mass spectroscopy. Additionally, we have observed that MppQ has a relatively high affinity for 2-ketoarginine, a shunt product in the L-End biosynthetic pathway, indicating a possible role of MppQ in increasing efficiency of L-End biosynthesis by converting 2-ketoarginine back to the starting material, L-arginine.

biochemistry↗

bioRxiv2022

Engineering a more specific E. coli glyoxylate/hydroxypyruvate reductase for coupled steady state kinetics assays

The E. coli glyoxylate reductase/hydroxypyruvate reductase A (EcGhrA) was investigated as a coupling enzyme to monitor the transamination of 2-ketoarginine and glycine by the L-enduracididine biosynthetic enzyme MppQ. Surprisingly, 2-ketoarginine proved to be an efficient substrate for EcGhrA. Since the promiscuity of EcGhrA prevented its use as a coupling enzyme to monitor the aminotransferase activity of MppQ, we set about engineering a more specific variant. X-ray crystal structures of EcGhrA were determined in the unliganded state, as well as with glyoxylate and 2-ketoarginine bound. The electron density maps of EcGhrA with 2-ketoarginine bound showed weak electron density for the side chain of this substrate, complicating the choice of active site residues to target for site-directed mutagenesis. The structure of the complex did, however, suggest that the side chain of W45 could interact with the guanidinium group of 2-ketoarginine. We therefore generated the EcGhrAW45F variant and tested it for activity with 2-ketoarginine, glyoxylate, oxaloacetate, -ketoglutarate, -oxofuranacetic acid, phenyl pyruvate, 3-mercaptopyruvate and 2-ketobutyric acid. The W45F variant exhibited a [~]10-fold decrease in the specificity constant (kcat/KM) for 2-ketoarginine, while the reaction with glyoxylate was not significantly impaired. The reactions of the W45F variant with the alternative substrates oxaloacetate and -ketoglutarate were also impaired. Thus, the W45F variant is a less promiscuous enzyme than the wild-type. This engineered EcGhrAW45F variant could be generally useful as a coupling system for enzymes that produce glyoxylate, such as 4-hydroxy-2-oxoglutarate aldolase or isocitrate lyase.

biochemistry↗