MOLECULAR BIOLOGICAL ANALYSIS OF A BIDIRECTIONAL HYDROGENASE FROM CYANOBACTERIA

An 8.9-kb segment with hydrogenase genes from the cyanobacterium Anaba ena variabilis has been cloned and sequenced. The sequences show homol ogy to the methyl-viologen-reducing hydrogenases from archaebacteria a nd, even more striking, to the NAD(+)-reducing enzymes from Alcaligene s eutrophus and Nocardia opaca as well as to the NADP(+)-dependent pro tein from Desulfovibrio fructosovorans. The cluster from A. variabilis contains genes coding for both the hydrogenase heterodimer (hoxH and hoxY) and for the diaphorase moiety (hoxU and hoxF) described for the A. eutrophus enzyme. In A. variabilis the gene cluster is split by two open reading frames (between hoxY and hoxH and between hoxU and hoxY respectively), and a probably non-coding 0.9-kb segment in an unusual way. The hoxH partial sequence from Anabaena 7119 and Anacystis nidula ns was amplified by PCR. Using the labeled segment from A. 7119 as pro be, Southern analysis revealed homologous gene segments in the cyanoba cteria A. 7119, Anabaena cylindrica, Anacystis nidulans and A. variabi lis. The bidirectional hydrogenase from A. nidulans was purified and d igests were sequenced. The amino acid sequences obtained showed partia l identities to the amino acid sequences deduced from the DNA data of the 8.9-kb segment from A. variabilis. Therefore the 8.9-kb segment co ntains the genes coding for the bidirectional, reversible hydrogenase from cyanobacteria. Crude extracts from A. nidulans perform NAD(P)H-de pendent H-2 evolution corroborating the molecular biological demonstra tion of the NAD(P)(+)-dependent hydrogenase in cyanobacteria.