Biochemical characterization of histidinol dehydrogenase from the human pathogen Neisseriagonorrhoeae.

Histidinol dehydrogenase (HisD, E.C. 1.1.1.23), encoded by the hisD gene and catalyzing the final two steps in the l-histidine biosynthesis, has emerged as a promising antibacterial target for several human pathogens, such as Brucella suis and Mycobacterium tuberculosis. Herein, biochemical properties on recombinant HisD from the human pathogen Neisseria gonorrhoeae (NgHisD) were characterized in detail. SDS-PAGE and size-exclusion chromatography revealed that recombinant NgHisD is a homodimer (∼105 kDa native size; ∼47 kDa subunit). Kinetic analysis identified this protein as a highly selective NAD+-dependent enzyme (specific activity: 17.3 ± 0.4 U mg-1), exhibiting minimal activity with NADP+. The KM values for NAD+ and l-histidinol were 0.9 ± 0.3 mM and 45 ± 6 μM, respectively. Maximal activity occurred at pH 9.5 and 45 °C in the presence of Mn2+. Heat-inactivation experiment showed that rapid inactivation began to occur following incubation at 45 °C for 30 min. The metal ions coordinated by the enzyme were not sequestered by 10 mM EDTA but were efficiently chelated by 1,10-phenanthroline. The recombinant NgHisD lost 85 % activity in the presence of 1 mM 1,10-phenantroline. Our study not only establishes a foundation for investigating the physiological function of NgHisD in vivo but also uncovers the absence of the canonical histidine biosynthesis pathway in Neisseria cinerea.