Abstract
Soluble pyridine nucleotide transhydrogenase (STH) transfers hydride between NADH and NADPH to maintain redox balance. In the present study, the sth gene from Gram-positive bacterium Streptomyces avermitilis (SaSTH) was expressed in Escherichia coli, and the recombinant STH protein was purified to homogeneity. Activity assays indicated that SaSTH was able to catalyze transhydrogenase reactions by using NADH or NADPH as reductants and thio-NAD+ as an oxidant. The apparent Km value for NADPH (74.5μM) was lower than that for NADH (104.0μM) and the apparent kcat/Km for NADPH (2704.7mM-1s-1) was higher than that for NADH (1129.8mM-1s-1). SaSTH showed optimal activity at 25°C and at a pH of 6.2. Heat-inactivation studies revealed that SaSTH remained stable below 55°C and that approximately 50% activity was preserved at 57°C for 20min. Analyses also showed that SaSTH activity was inhibited by divalent ions, particularly Co2+, Ni2+, and Zn2+. In addition, the transhydrogenase activity of SaSTH was inhibited by ATP and strongly stimulated by ADP and AMP. In summary, we characterized a recombinant enzyme exhibiting STH activity from Gram-positive bacteria for the first time. Our findings provide new options for cofactor engineering and industrial biocatalytic processes.
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