HypB protein of Bradyrhizobium japonicum is a metal-binding GTPase capable of binding 18 divalent nickel ions per dimer.

Abstract

Bradyrhizobium japonicum hypB encodes a protein containing an extremely histidine-rich region (24 histidine residues within a 39-amino-acid stretch) and guanine nucleotide-binding domains. The product of the hypB gene was overexpressed in Escherichia coli and purified by Ni2+-charged metal chelate affinity chromatography (MCAC) in a single step. In SDS/PAGE, HypB migrated at 38 kDaslightly larger than the calculated molecular mass (32.8 kDa). Purified HypB has GTPase activity with a kcat of 0.18 min-1 and a K. for GTP of 7 ,iM, and it has dGTPase activity as well.HypB exists as a dimer of molecular mass 78 kDa in native solution as determined by fast protein liquid chromatography on Superose 12. It binds 9.0 + 0.14 divalent nickel ions per monomer (18 Ni2+ per dimer) with aKd of 2.3 ,uM; it also binds Zn2+, Cu2+, Co2+, Cd2+, and Mn2+. In-frame deletion of the histidine-rich region (deletion of 38 amino acids including 23 histidine residues) resulted in a truncated HypB that did not bind to the MCAC column, whereas in-frame deletion of 14 amino acids including 8 histidine residues within HypB resulted in a truncated HypB that still bound to the column. The results indicate that the histidine residues within the histidine-rich region of HypB are involved in metal binding.Nitrogen-fixing prokaryotes have the ability to reduce atmospheric N2 to NH3, termed biological N2 fixation. Nitrogen fixation by rhizobia, in association with leguminous plants (such as alfalfa, clover, beans, peas, and soybeans), occurs in the specialized nodules on the plant roots; this allows the plants to grow well without added expensive nitrogenous fertilizers. The reduction of N2 to NH3 is catalyzed by the metalloenzyme nitrogenase. The nitrogenase reaction is very energy intensive, usually requiring between 16 and 42 ATP molecules per N2 reduced. In addition to reduction of N2 to form NH3, nitrogenases from all known sources catalyze an ATP-dependent H2 evolution reaction, and this H2 production is an inherent property of the nitrogenase reaction. About 25-35% of the total energy required for the nitrogenase reaction is committed to reduce protons to form H2 (1), representing a significant loss of energy. However, some N2-fixing symbionts possess an uptake hydrogenase that is capable of oxidizing the nitrogenase-evolved hydrogen. This process thereby regenerates ATP via a membrane-bound respiratory electron-transport chain and makes the overall nitrogen-fixing process more energy efficient. Moreover, the scavenging of 02 via H2 oxidation prevents 02-mediated damage to nitrogenase.Bradyrhizobium japonicum, the N2-fixing symbiont of soybean plants, synthesizes a Ni2+-containing uptake-type hydrogenase. The purified enzyme contains two subunits (2, 3), and the structural genes for hydrogenase have been sequenced (4).

Mycobacterial recA is cotranscribed with a potential regulatory gene called recX

“…The M. smegmatis orf1 product includes a histidine-rich region with potential nickel binding capacity in common with several other related proteins involved in processing nickel-containing enzymes; HypB of E. coli does not contain such a region, however. The histidine-rich region of Bradyrhizoboum japonicum HypB has been shown to function in nickel ion binding by deletion analysis (Fu et al, 1995). HypB of E. coli has also been shown to be a GTP-binding protein (Maier et al, 1993) and the four conserved domains of G-proteins (Bourne et al, 1991) identified; similar sequences are also found in the orf1 gene product although a threonine in the G-2 region thought to be critical is absent.…”

hyp Gene Products in Alcaligenes Eutrophus are part of a Hydrogenase‐Maturation System

“…In most cases, including A. eulrophus but not E. c d i , HypB proteins contain extended histidine clusters. Analyses of purified HypB of' Hradyrhizobiumjuponicum (Fu et al, 1995) and K. leguminosarum (Rey et al, 1994) revealed Ni binding to these clusters with a stoichiometry of 9 Ni and 4 Ni to each HypB monomcr, respectively. Much less is known on the function of the other hyp products.…”

Molecular Biology of Microbial Hydrogenases

“…CP: carbamoyl phosphate. present at the N terminus of HypB enables the protein to store nickel (Rey et al, 1994;Fu et al, 1995;Olson et al, 1997, Olson andMaier, 2000). In other organisms, such as E. coli or R. eutropha, the absence of this nickelstorage function has been assumed to be compensated by a highly effi cient nickel transport system (Eitinger and Mandrand-Berthelot, 2000).…”