CHARACTERIZATION OF AN OPERON ENCODING AN NADP-REDUCING HYDROGENASE IN DESULFOVIBRIO FRUCTOSOVORANS

A genomic DNA fragment from Desulfovibrio fructosovorans, which strong ly hybridized with the hydAB genes from Desulfovibrio vulgaris Hildenb orough, was cloned and sequenced. This fragment was found to contain f our genes, named hnd4, hndB, hndC, and hndD. Analysis of the sequence homologies indicated that HndA shows 29, 21, and 26% identity with the 24-kDa subunit from Bos taunts complex I, the 25 kDa subunit from Par acoccus denitrificans NADH dehydrogenase type I, and the N-terminal do main of HoxF subunit of the NAD-reducing hydrogenase from Alcaligenes eutrophus, respectively. HndB does not show any significant homology w ith any known protein. HndC shows 37 and 33% identity with the C-termi nal domain of HoxF and the 51-kDa subunit from B. taurus complex I, re spectively, and has the requisite structural features to be able to bi nd one flavin mononucleotide, one NAD, and three [4Fe-4S] clusters. Hn dD has 40, 42, and 48% identity with hydrogenase I from Clostridium pa steurianum and HydC and HydA from D. vulgaris Hildenborough, respectiv ely. The 4.5-kb length of the transcripts expressed in D. fructosovora ns and in Escherichia coli (pSS13) indicated that all four genes were present on the same transcription unit. The sizes of the four polypept ides were measured by performing heterologous expression of hndABCD in E. coli, using the T7 promoter/polymerase system. The products of hnd A, hndB, hndC, and hndD were 18.8, 13.8, 52, and 63.4 kDa, respectivel y. One hndC deletion mutant, called SM3, was constructed by performing marker exchange mutagenesis. Immunoblotting studies carried out on ce ll extracts from D. fructosovorans wild-type and SM3 strains, using an tibodies directed against HndC, indicated that the 52-kDa protein was recognized in extracts from the wild-type strain only. In soluble extr acts from D. fructosovorans wild type, a 10-fold induction of NADP red uction was observed when H. was present, but no H-2-dependent NAD redu ction ever occurred. This H-2-dependent NADP reductase activity disapp eared completely in extracts from SM3. These results indicate that the hnd operon actually encodes an NADP-reducing hydrogenase in D. fructo sovorans.