The hydrogenase gene cluster of Rhizobium leguminosarum bv. viciae contains an additional gene (hypX), which encodes a protein with sequence similarity to the N10-formyltetrahydrofolate-dependent enzyme family and is required for nickel-dependent hydrogenase processing and activity.

Plasmid pAL618 contains the genetic determinants for H2 uptake (hup) from Rhizobium leguminosarum bv. viciae, including a cluster of 17 genes named hupSLCDEFGHIJK-hypABFCDE. A 1.7-kb segment of insert DNA located downstream of hypE has now been sequenced, thus completing the sequence of the 20441-bp insert DNA in plasmid pAL618. An open reading frame (designated hypX) encoding a protein with a calculated M(r) of 62300 that exhibits extensive sequence similarity with HoxX from Alcaligenes eutrophus (52% identity) and Bradyrhizobium japonicum (57% identity) was identified 10 bp downstream of hypE. Nodule bacteroids produced by hypX mutants in pea (Pisum sativum L.) plants grown at optimal nickel concentrations (100 microM) for hydrogenase expression, exhibited less than 5% of the wild-type levels of hydrogenase activity. These bacteroids contained wild-type levels of mRNA from hydrogenase structural genes (hupSL) but accumulated large amounts of the immature form of HupL protein. The Hup-deficient mutants were complemented for normal hydrogenase activity and nickel-dependent maturation of HupL by a hypX gene provided in trans. From expression analysis of hypX-lacZ fusion genes, it appears that hypX gene is transcribed from the FnrN-dependent hyp promoter, thus placing hypX in the hyp operon (hypBFCDEX). Comparisons of the HypX/HoxX sequences with those in databases provided unexpected insights into their function in hydrogenase synthesis. Similarities were restricted to two distinct regions in the HypX/HoxX sequences. Region I, corresponding to a sequence conserved in N10-formyltetrahydrofolate-dependent enzymes involved in transferring one-carbon units (C1), was located in the N-terminal half of the protein, whereas region II, corresponding to a sequence conserved in enzymes of the enoyl-CoA hydratase/isomerase family, was located in the C-terminal half. These similarities strongly suggest that HypX/HoxX have dual functions: binding of the C1 donor N10-formyltetrahydrofolate and transfer of the C1 to an unknown substrate, and catalysis of a reaction involving polarization of the C = O bond of an X-CO-SCoA substrate. These results also suggest the involvement of a small organic molecule, possibly synthesized with the participation of an X-CO-SCoA precursor and of formyl groups, in the synthesis of the metal-containing active centre of hydrogenase.