Biochemical Characterization of Homoserine Dehydrogenase from Pseudomonas aeruginosa

Abstract

Homoserine dehydrogenase (HSD) catalyzes the reduction of l‐aspatate‐4‐semaldehyde (l‐ASA) to l‐homoserine (l‐HSE) or oxidation reversely, which is a part of the aspartate pathway synthesizing threonine, isoleucine, and methionine in vivo. HSD has gained much interest in medical application since HSD is a well‐established target for pesticides and antibiotics. In addition, HSD is also valuable in industrial application for l‐lysine production. In this study, HSD from Pseudomonas aeruginosa (PaHSD) was overexpressed in Escherichia coli, and purified to homogeneity for molecular and biochemical characterization. PaHSD exhibits the comparable activity of l‐HSE oxidation for both types of cofactors, NAD+ and NADP+, but at the same time shows interesting differences. The kcat/Km value of NADP+ was ~1.8 times larger than that of NAD+, while the kcat/Km value of l‐HSE was ~1.4 times larger with NAD+ than with NADP+. Notably, the Km value is about three times smaller for NADP+, while Vmax is about 1.7 times larger for NAD+, which implies that while NADP+ binds more strongly to the active site, NAD+ has a faster turnover. Size‐exclusion chromatography analysis showed PaHSD forms tetramers, as opposed to HSDs from other species forming dimers. In the circular dichroism analysis, PaHSD showed a typical spectrum of α/β structure, and its melting temperature was determined to be 50.5 °C from the thermal denaturation test at 222 nm. The molecular and biochemical features of PaHSD revealed in this study provide the basis for the future study of amino acid metabolism, and its application to industrial and medical purposes.

Comparative Proteomic Analysis for a Putative Pyridoxal Phosphate-Dependent Aminotransferase Required for Virulence in Acidovorax citrulli

“…In this study, we observed that glutamate 5-kinase (ATG93720), which is involved in glutamate synthesis, was more abundant in the wild-type than in the mutant. In biochemical pathways, glutamates are converted to aspartates, which, in turn, can be degraded to homoserine by homoserine dehydrogenase; the homoserines can be further converted to methionine and threonine ( Kim et al, 2020a ). Based upon our data, the enzymes necessary for the above-mentioned biochemical pathways, homoserine dehydrogenase (ATG95108), threonine synthase (ATG95107), and methionine synthase (ATG95998), are more abundant in the wild-type than in the mutant.…”