Biochemical characterization of histidinol dehydrogenase from the human pathogen Neisseria gonorrhoeae

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 ( Ng HisD) were characterized in detail. SDS-PAGE and size-exclusion chromatography revealed that recombinant Ng HisD 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 K M 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 Mn 2+ . 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 Ng HisD 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 Ng HisD in vivo but also uncovers the absence of the canonical histidine biosynthesis pathway in Neisseria cinerea .