HypB protein of Bradyrhizobium japonicum is a metal-binding GTPase capable of binding 18 divalent nickel ions per dimer.
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 Ni(2+)-charged metal chelate affinity chromatography (MCAC) in a single step. In SDS/PAGE, HypB migrated at 38 kDa--slightly larger than the calculated molecular mass (32.8 kDa). Purified HypB has GTPase activity with a kcat of 0.18 min-1 and a Km for GTP of 7 microM, 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 a Kd of 2.3 microM; 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.
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Department of Biology, Johns Hopkins University, Baltimore, MD 21218.
Olson
J W
JW
Maier
R J
RJ
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D006639:Histidine; D007700:Kinetics; D046911:Macromolecular Substances; D016296:Mutagenesis; D009532:Nickel / Q000378:metabolism*; D016364:Reading Frames; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012230:Rhizobiaceae / Q000378:metabolism*; D017384:Sequence Deletion
D001426:Bacterial Proteins; D002352:Carrier Proteins; D002413:Cations, Divalent; D029968:Escherichia coli Proteins; C490324:HypB protein, E coli; C089637:HypB protein, bacteria; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D006639:Histidine; D009532:Nickel; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins