Carboxyl-terminal processing of the cytoplasmic NAD-reducing hydrogenase of Alcaligenes eutrophus requires the hoxW gene product

Abstract

Two open reading frames (ORFs) were identified immediately downstream of the four structural genes for the soluble hydrogenase (SH) of Alcaligenes eutrophus H16. While a mutation in ORF2 had no obvious effect on hydrogen metabolism, an in-frame deletion in ORF1, subsequently designated hoxW, led to a complete loss of SH activity and hence a significant retardation of autotrophic growth on hydrogen. Hydrogen oxidation in the hoxW mutant was catalyzed by the second hydrogenase, a membrane-bound enzyme. Assembly of the four subunits of the SH was blocked in mutant cells, and HoxH, the hydrogen-activating subunit, accumulated as a precursor which was still capable of binding nickel. Protein sequencing revealed that HoxH isolated from the wild type terminates at His-464, whereas the C-terminal amino acid sequence of HoxH from the hoxW mutant is colinear with the deduced sequence. Processing of the HoxH precursor was restored in vitro by a cell extract containing HoxW. These results indicate that HoxW is a highly specific carboxyl-terminal protease which releases a 24-amino-acid peptide from HoxH prior to progression of subunit assembly.The facultative lithoautotroph Alcaligenes eutrophus can use molecular hydrogen as the sole energy source. Hydrogen oxidation in this proteobacterium is mediated by two (NiFe)-containing hydrogenases. The membrane-bound hydrogenase (MBH) is composed of a large and a small subunit, which are coupled to electron transport phosphorylation via a cytochrome b-like anchor protein. While the MBH is a representative of the more common type of (NiFe)-hydrogenases, the second, soluble hydrogenase (SH) belongs to a family of less abundant multimeric (NiFe)-hydrogenases (reviewed in reference 7). Multimeric hydrogenases are present in a few Alcaligenes species, in the gram-positive Rhodococcus sp. strain 1b (formerly Nocardia opaca) (4), and in the cyanobacterium Anabaena variabilis (32).The cytoplasmic SH reacts with NAD as the physiological electron carrier. The enzyme is composed of four heterologous subunits (35) encoded by the megaplasmid-borne genes hoxF, hoxU, hoxY, and hoxH (39). The products of hoxF and hoxU form the flavononheme iron protein of the SH which possesses NADH oxidoreductase (diaphorase) activity (33). The diaphorase dimer is closely related to three peripheral subunits of the NADH:ubiquinone oxidoreductase (complex I) of mitochondria and of some bacterial species (42, 43). The hoxH and hoxY gene products show features of a hydrogen-activating site and an intramolecular electron transfer pathway (7). The first crystallographic analysis of a (NiFe)-hydrogenase carried out for the periplasmic hydrogenase of Desulfovibrio gigas was recently completed. According to this study, the active site appears to be a binuclear cluster of nickel and probably iron, buried deep inside the protein (41).Genetic studies on various bacteria uncovered a series of accessory genes which code for functions involved in the biogenesis of (NiFe)-hydrogenases. Several specific gene products are required...

Probing the Origin of the Metabolic Precursor of the CO Ligand in the Catalytic Center of [NiFe] Hydrogenase

“…It is anticipated that the resulting cofactor intermediate is subsequently transferred to the apo-hydrogenase (13, 18 -20). In a process assisted by the HypA and HypB proteins, nickel is inserted (21), and maturation of the active site-containing large subunit is completed through specific endoproteolytic cleavage of a C-terminal amino acid extension that triggers protein folding and the final assembly with the electron-transferring small subunit (22,23 3 spectroscopy revealed that 13 CO, externally supplied to hydrogenase-synthesizing cell cultures, is efficiently incorporated into the active site of the enzymes (12,14). Taking an average CO concentration of 0.1 ppmv (24) in the northern hemisphere into account, it is theoretically conceivable that atmospheric CO serves as the source of the carbonyl ligand.…”

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

“…Near the carboxy terminus, HoxH possesses a strongly conserved motif ending with a histidine. The formation of the mature protein and the incorporation of Ni into it might require the cleavage of a stretch of 26 amino acids behind this His similar as in the proteins from Methanococcus voltae [16], E. eoli (hydrogenase 3) [17], and A. eutrophus [18]. An N-terminal leader sequence is also not observed in HoxH of A. nidulans.…”

“…1, Table 1). A sequence for a protein with no obvious function (ORF2) resides between hoxW and hypA in A. eutrophus [18]. Such a protein is not encoded in the same region on the A. nidulans genome.…”

“…Such a low value is not surprising, since these proteases appear to cleave stretches specifically from distinct hydrogenases. The motif GxGNx4DD/EGxG, common in proteases cleaving the hydrogenase large subunit [18], occurs in HoxW of A. nidulans also.…”

“…The protein encoded by hypF is believed to be involved in the H2 stimulation of hydrogenase expression. In many organisms with the exception of A. eutrophus [18] HypF possesses a Cys arrangement typical of a zinc-finger protein which is possibly present on the part not yet sequenced. As mentioned above, hypF is not located in the vicinity of other hydrogenase genes in Synechocystis 6803 [21], indicating either a different organization of the hydrogenase accessory genes in this cyanobacterium or the presence of a second copy of this gene.…”