HYPB PROTEIN OF BRADYRHIZOBIUM-JAPONICUM IS A METAL-BINDING GTPASE CAPABLE OF BINDING 18 DIVALENT NICKEL IONS PER DIMER

被引:102
作者
FU, CL
OLSON, JW
MAIER, RJ
机构
[1] Department of Biology, Johns Hopkins University, Baltimore
关键词
HYDROGENASE; METALLOPROTEIN; NITROGEN FIXATION;
D O I
10.1073/pnas.92.6.2333
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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 kDa-slightly larger than the calculated molecular mass (32.8 kDa). Purified HypB has GTPase activity with a k(cat) of 0.18 min(-1) and a K-m for GTP of 7 mu M, 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 K-d of 2.3 mu M; 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.
引用
收藏
页码:2333 / 2337
页数:5
相关论文
共 31 条
[1]  
AMES BN, 1960, J BIOL CHEM, V235, P769
[3]   SEQUENCES AND CHARACTERIZATION OF HUPU AND HUPV GENES OF BRADYRHIZOBIUM-JAPONICUM ENCODING A POSSIBLE NICKEL-SENSING COMPLEX INVOLVED IN HYDROGENASE EXPRESSION [J].
BLACK, LK ;
FU, CL ;
MAIER, RJ .
JOURNAL OF BACTERIOLOGY, 1994, 176 (22) :7102-7106
[4]  
BOURNE HR, 1991, NATURE, V349, P117, DOI 10.1038/349117a0
[5]   IDENTIFICATION OF 6 OPEN READING FRAMES FROM A REGION OF THE AZOTOBACTER-VINELANDII GENOME LIKELY INVOLVED IN DIHYDROGEN METABOLISM [J].
CHEN, JC ;
MORTENSON, LE .
BIOCHIMICA ET BIOPHYSICA ACTA, 1992, 1131 (02) :199-202
[6]   ORGANIZATION OF THE GENES NECESSARY FOR HYDROGENASE EXPRESSION IN RHODOBACTER-CAPSULATUS - SEQUENCE-ANALYSIS AND IDENTIFICATION OF 2 HYP REGULATORY MUTANTS [J].
COLBEAU, A ;
RICHAUD, P ;
TOUSSAINT, B ;
CABALLERO, FJ ;
ELSTER, C ;
DELPHIN, C ;
SMITH, RL ;
CHABERT, J ;
VIGNAIS, PM .
MOLECULAR MICROBIOLOGY, 1993, 8 (01) :15-29
[7]   ANALYSIS OF A PLEIOTROPIC GENE REGION INVOLVED IN FORMATION OF CATALYTICALLY ACTIVE HYDROGENASES IN ALCALIGENES-EUTROPHUS H16 [J].
DERNEDDE, J ;
EITINGER, M ;
FRIEDRICH, B .
ARCHIVES OF MICROBIOLOGY, 1993, 159 (06) :545-553
[8]   ASPECTS OF HYDROGEN METABOLISM IN NITROGEN-FIXING LEGUMES AND OTHER PLANT-MICROBE ASSOCIATIONS [J].
EISBRENNER, G ;
EVANS, HJ .
ANNUAL REVIEW OF PLANT PHYSIOLOGY AND PLANT MOLECULAR BIOLOGY, 1983, 34 :105-136
[9]   HYDROGEN-UBIQUINONE OXIDOREDUCTASE ACTIVITY BY THE BRADYRHIZOBIUM-JAPONICUM MEMBRANE-BOUND HYDROGENASE [J].
FERBER, DM ;
MAIER, RJ .
FEMS MICROBIOLOGY LETTERS, 1993, 110 (03) :257-264
[10]  
FU CL, 1994, GENE, V145, P91, DOI 10.1016/0378-1119(94)90328-X