Comparison of alkyl hydroperoxide reductase and two water-forming NADH oxidases from Bacillus cereus ATCC 14579

Abstract

Bacillus cereus (B. cereus) is an ubiquitous facultative anaerobic bacterium, and its growth in aerobic environment correlates to the functions of its oxygen defense system. Water-forming NADH oxidase (nox-2) can catalyze the conversion of oxygen to water with concomitant NADH oxidation in anaerobic microorganisms. Here, we report the cloning and characterization of two annotated nox-2s (nox-2(444) and nox-2(554)) from B. cereus ATCC 14579 and their comparison with another oxidative stress defense system alkyl hydroperoxide reductase (AhpR) from this microbe, which composed of two enzymes-hydrogen peroxide-forming NADH oxidase (nox-1) and peroxidase. Both nox-2 and AhpR catalyze the same reaction in the presence of oxygen. With the stimulation of exogenously added FAD, the maximum activity of nox-1, nox-2(444), and nox-2(554) could reach 27.7 U/mg, 22.9 U/mg, and 2.4 U/mg, respectively, at pH 7.0, 30°C. Different from nox-1, both nox-2s were thermotolerant enzymes and could maintain above 87% of their optimum activity at 80°C, which was not found in other nox-2s. As for operational stability, all are turnover-limited. Exogenously added reductive reagent dithiothreitol could dramatically increase the total turnover number of nox-2(444) and nox-2(554) by twofold and threefold, respectively, but had no effect on AhpR or nox-1.