Gonorrhoea, caused by Neisseria gonorrhoeae, is a major global public health concern. Homoserine dehydrogenase (HSD), a key enzyme in the aspartate pathway, is a promising metabolic target against pathogenic infections. In this study, a monofunctional HSD from N. gonorrhoeae (NgHSD) was overexpressed in Escherichia coli and purified to >95% homogeneity for biochemical characterization. Unlike the classic dimeric structure, the purified recombinant NgHSD exists as a tetramer in solution. We determined the enzymatic activity of recombinant NgHSD for l-homoserine oxidation, which revealed that this enzyme was NAD+ dependent, with an approximate 479-fold (kcat/Km) preference for NAD+ over NADP+, and that optimal activity for l-homoserine oxidation occurred at pH 10.5 and 40 °C. At 800 mM, neither NaCl nor KCl increased the activity of NgHSD, in contrast to the behavior of several reported NAD+-independent homologs. Moreover, threonine did not markedly inhibit the oxidation activity of NgHSD. To gain insight into the cofactor specificity, site-directed mutagenesis was used to alter coenzyme specificity. The double mutant L45R/S46R, showing the highest affinity for NADP+, caused a shift in coenzyme preference from NAD+ to NADP+ by a factor of ~974, with a catalytic efficiency comparable with naturally occurring NAD+-independent homologs. Collectively, our results should allow the exploration of drugs targeting NgHSD to treat gonococcal infections and contribute to the prediction of the coenzyme specificity of novel HSDs.
Keywords: Affinity purification; Biochemical properties; Gonococcus; Homoserine dehydrogenase; Site-directed mutagenesis.
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