title: Molecular characterization of a glucokinase with broad hexose specificity from Bacillus sphaericus strain C3-41.
abstract: Bacillus sphaericus cannot metabolize sugar since it lacks several of the enzymes necessary for glycolysis. Our results confirmed the presence of a glucokinase-encoding gene, glcK, and a phosphofructokinase-encoding gene, pfk, on the bacterial chromosome and expression of glucokinase during vegetative growth of B. sphaericus strains. However, no phosphoglucose isomerase gene (pgi) or phosphoglucose isomerase enzyme activity was detected in these strains. Furthermore, one glcK open reading frame was cloned from B. sphaericus strain C3-41 and then expressed in Escherichia coli. Biochemical analysis revealed that this gene encoded a protein with a molecular mass of 33 kDa and that the purified recombinant glucokinase had K(m) values of 0.52 and 0.31 mM for ATP and glucose, respectively. It has been proved that this ATP-dependent glucokinase can also phosphorylate fructose and mannose, and sequence alignment of the glcK gene indicated that it belongs to the ROK protein family. It is postulated that the absence of the phosphoglucose isomerase-encoding gene pgi in B. sphaericus might be one of the reasons for the inability of this bacterium to metabolize carbohydrates. Our findings provide additional data that further elucidate the specific metabolic pathway and could be used for genetic improvement of B. sphaericus.
authors.affiliation: Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
authors.forename: Bei
authors.initials: B
authors.lastname: Han
authors.forename: Haizhou
authors.initials: H
authors.lastname: Liu
authors.forename: Xiaomin
authors.initials: X
authors.lastname: Hu
authors.forename: Yajun
authors.initials: Y
authors.lastname: Cai
authors.forename: Dasheng
authors.initials: D
authors.lastname: Zheng
authors.forename: Zhiming
authors.initials: Z
authors.lastname: Yuan
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D002876:Chromosomes, Bacterial / Q000235:genetics; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005632:Fructose / Q000378:metabolism; D015870:Gene Expression; D005941:Glucokinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D005956:Glucose-6-Phosphate Isomerase / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008358:Mannose / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010732:Phosphofructokinase-1 / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D005632:Fructose; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1; D005941:Glucokinase; D005956:Glucose-6-Phosphate Isomerase; D005947:Glucose; D008358:Mannose