Molecular Biological Analysis of a Bidirectional Hydrogenase from Cyanobacteria

Abstract

An 8.9-kb segment with hydrogenase genes from the cyanobacterium Anabaena variabilis has been cloned and sequenced. The sequences show homology to the methyl-viologen-reducing hydrogenases from archaebacteria and, even more striking, to the NAD'-reducing enzymes from Alcaligenes eutrophus and Nocardia opaca as well as to the NADP' -dependent protein from Desulfovibrio fructosovorarzs. The cluster from A. variabilis contains genes coding for both the hydrogenase heterodimer (hoxH and hoxv and for the diaphorase moiety (hoxU and hoxfl described for the A. eurroplius enzyme. In A. variabilis the gene cluster is split by two open reading frames (between hoxY and hoxH and between hoxU and hoxl: respectively), and a probably non-coding 0.9-kb segment in an unusual way. The hoxH partial sequence from Anabaena 71 19 and Anucystis nidulan.7 was amplified by PCR. Using the labeled segment from A. 71 19 as probe, Southern analysis revealed homologous gene segments in the cyanobacteria A. 71 19, Anabaena cylindrica, Arzacystis nidulans and A. variabilis. The bidirectional hydrogenase from A. nidulans was purified and digests were sequenced. The amino acid sequences obtained showed partial 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 contains the genes coding for the bidirectional, reversible hydrogenase from cyanobacteria. Crude extracts from A. nidulans perform NAD(P)H-dependent H, evolution corroborating the molecular biological demonstration of the NAD(P)'.-dependent hydrogenase in cyanobacteria.

Cloning, molecular analysis and insertional mutagenesis of the bidirectional hydrogenase genes from the cyanobacterium Anacystis nidulans

“…The deduced amino acid sequences of the hoxU, hoxY and hoxH genes all have the same sequence characteristics described for the homologous genes from A. variabilis [7], in particular the conserved cysteine motifs for binding potential Fe-S clusters and for the Ni-binding sites. The HoxU protein (the small subunit of the diaphorase part of the enzyme) possesses 238 amino acids with 16 cysteines (for comparison 238 amino acids with 15 Cys in HoxU of A. variabilis [7]).…”

“…Characterization of the structural genes of the bidirectional hydrogenase As described in a preceding publication, an 1.35 kb DNA segment from several cyanobacteria could be amplified by PCR with specific primers, and sequencing of this segment indicated its strong identity to part of the large subunit (hoxH gene) of an NAD+-reducing hydrogenase [7]. The digoxigenin-labeled DNA segment from Anabaena sp.…”

“…The genomic DNA bank of A. nidulans in X-GEMI1 was constructed as described for the A. variabilis bank [7]. Protein was determined by the Lowry method, and all chemicals came from Boehringer, Mannheim.…”

“…In the previous investigation [7], the bidirectional hydrogenase from A. nidulans had been purified and digests had been sequenced. The determined amino acid sequence of a digest of the large subunit (hoxH gene product) was identical with that deduced from the DNA sequence in the case of A. nidulans and was strongly homologous to that deduced from the A. variabilis DNA composition, as shown in the table below.…”

“…In the case of A. variabilis, two open reading frames are located within the gene cluster (ORF3 between hoxY and hoxH and ORF8 between hoxU and hoxY," see Fig. 1 and [7]). ORF3 could not be detected on genomic DNA from A. nidulans by heterologous probing even at the low hybridization temperature of 56°C.…”