THE HYDROGENASE GENE-CLUSTER OF RHIZOBIUM-LEGUMINOSARUM BV. VICIAE CONTAINS AN ADDITIONAL GENE (HYPX), WHICH ENCODES A PROTEIN WITH SEQUENCE SIMILARITY TO THE N-10-FORMYLTETRAHYDRAFOLATE-DEPENDENT ENZYME FAMILY AND IS REQUIRED FOR NICKEL-DEPENDENT HYDROGENASE PROCESSING AND ACTIVITY

Plasmid pAL618 contains the genetic determinants for Hz uptake (hup) f rom Rhizobium leguminosarum by. viciae, including a cluster of 17 gene s named hupSLCDEFGHIJK-hypABFCDE. A 1.7-kb segment of insert DNA locat ed downstream of hypE has now been sequenced, thus completing the sequ ence of the 20441-bp insert DNA in plasmid pAL618. An open reading fra me (designated hypX) encoding a protein with a calculated M(r) of 62 3 00 that exhibits extensive sequence similarity with HoxX from Alcalige nes eutrophus (52% identity) and Bradyrhizobium japonicum (57% identit y) was identified 10 bp downstream of hypE. Nodule bacteroids produced by hypX mutants in pea (Pisum sativum L.) plants grown at optimal nic kel concentrations (100 mu M) for hydrogenase expression, exhibited le ss than 5% of the wild-type levels of hydrogenase activity. These bact eroids contained wild-type levels of mRNA from hydrogenase structural genes (hupSL) but accumulated large amounts of the immature form of Hu pL protein. The Hup-deficient mutants were complemented for normal hyd rogenase activity and nickel-dependent maturation of HupL by a hypX ge ne provided in trans. From expression analysis of hypX-lacZ fusion gen es, it appears that hypX gene is transcribed from the FnrN-dependent h yp promoter, thus placing hypX in the hyp operon (hypBFCDEX). Comparis ons of the HypX/HoxX sequences with those in databases provided unexpe cted insights into their function in hydrogenase synthesis. Similariti es were restricted to two distinct regions in the HypX/HoxX sequences. Region I, corresponding to a sequence conserved in N-10-formyltetrahy drofolate-dependent enzymes involved in transferring one-carbon units (C-1), was located in the N-terminal half of the protein, whereas regi on II, corresponding to a sequence conserved in enzymes of the enoyl-C oA hydratase/isomerase family, was located in the C-terminal half, The se similarities strongly suggest that HypX/HoxX have dual functions: b inding of the C-1 donor N-10-formyltetrahydrofolate and transfer of th e C-1 to an unknown substrate, and catalysis of a reaction involving p olarization of the C=O bond of an X-CO-SCoA substrate. These results a lso suggest the involvement of a small organic molecule, possibly synt hesized with the participation of an X-CO-SCoA precursor and of formyl groups, in the synthesis of the metal-containing active centre of hyd rogenase.