Dual roles of Bradyrhizobium japonicum nickelin protein in nickel storage and GTP-dependent Ni mobilization.

The maturation of nickel-containing enzymes, involving poorly described steps of nickel mobilization and insertion into metal centers, has been the subject of increasing scrutiny over the last few years with the sequencing of genes encoding “accessory” proteins required for Ni-containing-enzyme synthesis (see reference 9). For the three best understood systems—hydrogenase, urease, and carbon monoxide dehydrogenase (CODH)—interesting parallels have emerged between the properties of these accessory proteins (10). In each system, there appears to be a requirement for an accessory protein with a nucleotide-binding motif. This motif is proposed to function in a chaperone-type role for synthesis of active-site metallocenters in urease, hydrogenase, nitrous oxide reductase, and nitrogenase (9). Also conserved to varying degrees among the nickel enzymes are accessory proteins with histidine-rich areas, which in some cases have been shown to be the domains that bind nickel. Several proteins have been shown to be required for urease metallocenter biosynthesis. One of these is the histidine-rich protein UreE (Fig. ​(Fig.1A),1A), and another is the nucleotide-binding protein UreG (15, 16). Similarly, CODH maturation requires the histidine-rich protein CooJ (Fig. ​(Fig.1A)1A) and the nucleotide-binding protein CooC (12). FIG. 1 Alignments of the histidine-rich regions of known or putative HypB proteins from various organisms, including the histidine-rich regions of UreE and CooJ (A), and the G motifs from several known or putative HypB proteins (B). Also included are the nucleotide-binding ... In the case of hydrogenase maturation systems, the two properties (nickel sequestering and nucleotide binding/hydrolysis) can be contained in a single protein, namely, HypB (8, 23). Bradyrhizobium japonicum HypB purified from an over-producing strain of Escherichia coli has been shown to bind up to 18 nickel ions per dimer and also to contain GTPase activity (8). In-frame mutations of hypB yield strains which are partially or completely deficient in hydrogenase activity, depending on how much of the gene is deleted. A strain which produces a truncated HypB lacking 23 of the clustered 24 histidines is still capable of producing active hydrogenase, but these activities only approach wild-type levels when very high levels of nickel are supplied to the growth medium (23). The strain expressing the truncated hypB also accumulates less nickel than the wild type under conditions when hypB is expressed (23). From these results, we have concluded that HypB has two roles in B. japonicum: (i) that of nickel binding and storage, with this function being dependent on the histidine-rich N terminus, and (ii) that of hydrogenase expression, which may require the nucleotide-binding motif and GTP hydrolysis. Due to the nickel storage role, we previously proposed the name “nickelin” for HypB (J. W. Olson, C. Fu, and R. J. Maier, Abstr. 96th Gen. Meet. Am. Soc. Microbiol. 1996, abstr. K-202, p. 570, 1996). Here we report that these two functions of HypB can be separated and assigned to separate domains of the protein. In vitro analysis of a truncated form of the protein missing 23 of the 24 clustered histidines shows that it retains the properties required for hydrogenase synthesis, while a mutation in the G1 domain of nickelin demonstrates that GTP hydrolysis is essential for nickelin's role in nickel donation to form an active hydrogenase.