Accessory proteins functioning selectively and pleiotropically in the biosynthesis of [NiFe] hydrogenases in Thiocapsa roseopersicina

Abstract

There are at least two membrane-bound (HynSL and HupSL) and one soluble (HoxEFUYH) [NiFe] hydrogenases in Thiocapsa roseopersicina BBS, a purple sulfur photosynthetic bacterium. Genes coding for accessory proteins that participate in the biosynthesis and maturation of hydrogenases seem to be scattered along the chromosome. Transposon-based mutagenesis was used to locate the hydrogenase accessory genes. Molecular analysis of strains showing mutant phenotypes led to the identification of hupK (hoxV ), hypC 1 , hypC 2 , hypD, hypE, and hynD genes. The roles of hynD, hupK and the two hypC genes were investigated in detail. The putative HynD was found to be a hydrogenase-specific endoprotease type protein, participating in the maturation of the HynSL enzyme. HupK plays an important role in the formation of the functionally active membrane-bound [NiFe] hydrogenases, but not in the biosynthesis of the soluble enzyme. In-frame deletion mutagenesis showed that HypC proteins were not specific for the maturation of either hydrogenase enzyme. The lack of either HypC protein drastically reduced the activity of every hydrogenase. Hence both HypCs might participate in the maturation of [NiFe] hydrogenases. Homologous complementation with the appropriate genes substantiated the physiological roles of the corresponding gene products in the H 2 metabolism of T. roseopersicina.

Heterologous expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] hydrogenases in Escherichia coli

“…A previously published report showed lack of hydrogenase activity when the hynSL genes from T. roseopersicina were expressed in E. coli indicating that additional maturation proteins are required for proper maturation (Shirshikova et al, 2009). Because the genome sequence of T. roseopersicina has not yet been published, only a subset of the T. roseopersicina genes typically required for [NiFe] hydrogenase maturation have been discovered from mutational studies (Fodor et al, 2001;Maró ti et al, 2003). When we assembled these genes along with the hydrogenase structural genes into artificial operons and expressed them in E. coli, no activity was observed, suggesting that some essential T. roseopersicina accessory genes could be missing.…”

“…A number of T. roseopersicina genes encoding accessory proteins have been identified by mutagenesis including hynD, hupK, hypC1, hypC2, hypD, hypE and hypF (Fodor et al, 2001;Kovács et al, 2002). HynD is an endoprotease involved in cleaving the large subunit HynL, while HypCDEF are thought to function in assembly of the active [NiFe] catalytic site (Maró ti et al, 2003;Maró ti et al, 2009).…”

Discovery of [NiFe] Hydrogenase Genes in Metagenomic DNA: Cloning and Heterologous Expression in Thiocapsa roseopersicina

“…The origin of the CO ligand that is also bound to the iron is not clear, and possibly it comes from formate, formyl-tetrahydrofolate, or acetate. The liganded Fe atom is inserted into the immature large subunit, in which HypC proteins function as chaperones to facilitate the metal insertion (5,34,36). Ni is delivered to the catalytic center by the zinc-metalloenzyme HypA that interacts with HypB, a nickel-binding and GTP-hydrolyzing protein.…”

Cyanobacterial-Type, Heteropentameric, NAD + -Reducing NiFe Hydrogenase in the Purple Sulfur Photosynthetic Bacterium Thiocapsa roseopersicina

“…No hydrogenase accessory gene was found in the vicinity of hynSL, while downstream of hupSL several genes homologous to spe-cific accessory genes were identified (10). Seven genes coding for accessory proteins involved in the biosynthesis of hydrogenases were isolated after transposon mutagenesis in the genome of T. roseopersicina (13,24). These genes are clustered in various loci.…”