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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.

SUBMITTER: Han B

PROVIDER: S-EPMC1932687 | biostudies-literature | 2007 Jun

REPOSITORIES: biostudies-literature

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Molecular characterization of a glucokinase with broad hexose specificity from Bacillus sphaericus strain C3-41.

Han Bei B Liu Haizhou H Hu Xiaomin X Cai Yajun Y Zheng Dasheng D Yuan Zhiming Z

Applied and environmental microbiology 20070330 11

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 ...[more]

PMID: 17400775

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Project description:An ATP-dependent glucokinase of the hyperthermophilic aerobic crenarchaeon Aeropyrum pernix was purified 230-fold to homogeneity. The enzyme is a monomeric protein with an apparent molecular mass of about 36 kDa. The apparent K(m) values for ATP and glucose (at 90 degrees C and pH 6.2) were 0.42 and 0.044 mM, respectively; the apparent V(max) was about 35 U/mg. The enzyme was specific for ATP as a phosphoryl donor, but showed a broad spectrum for phosphoryl acceptors: in addition to glucose, which showed the highest catalytic efficiency (k(cat)/K(m)), the enzyme also phosphorylates glucosamin, fructose, mannose, and 2-deoxyglucose. Divalent cations were required for maximal activity: Mg(2+), which was most effective, could partially be replaced with Co(2+), Mn(2+), and Ni(2+). The enzyme had a temperature optimum of at least 100 degrees C and showed significant thermostability up to 100 degrees C. The coding function of open reading frame (ORF) APE2091 (Y. Kawarabayasi, Y. Hino, H. Horikawa, S. Yamazaki, Y. Haikawa, K. Jin-no, M. Takahashi, M. Sekine, S. Baba, A. Ankai, H. Kosugi, A. Hosoyama, S. Fukui, Y. Nagai, K. Nishijima, H. Nakazawa, M. Takamiya, S. Masuda, T. Funahashi, T. Tanaka, Y. Kudoh, J. Yamazaki, N. Kushida, A. Oguchi, and H. Kikuchi, DNA Res. 6:83-101, 145-152, 1999), previously annotated as gene glk, coding for ATP-glucokinase of A. pernix, was proved by functional expression in Escherichia coli. The purified recombinant ATP-dependent glucokinase showed a 5-kDa higher molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but almost identical kinetic and thermostability properties in comparison to the native enzyme purified from A. pernix. N-terminal amino acid sequence of the native enzyme revealed that the translation start codon is a GTG 171 bp downstream of the annotated start codon of ORF APE2091. The amino acid sequence deduced from the truncated ORF APE2091 revealed sequence similarity to members of the ROK family, which comprise bacterial sugar kinases and transcriptional repressors. This is the first report of the characterization of an ATP-dependent glucokinase from the domain of Archaea, which differs from its bacterial counterparts by its monomeric structure and its broad specificity for hexoses.

Project description:5'-nucleotidases (EC 3.1.3.5) catalyze the hydrolytic dephosphorylation of 5'-ribonucleotides and 5'-deoxyribonucleotides as well as complex nucleotides, such as uridine 5'-diphosphoglucose (UDP-glucose), nicotinamide adenine dinucleotide and flavin adenine dinucleotide, to their corresponding nucleosides plus phosphate. These enzymes have been found in diverse species in intracellular and membrane-bound, surface-localized forms. Soluble forms of 5'-nucleotidases belong to the ubiquitous haloacid dehalogenase superfamily (HADSF) and have been shown to be involved in the regulation of nucleotide, nucleoside and nicotinamide adenine dinucleotide (NAD+) pools. Despite the important role of 5'-nucleotidases in cellular metabolism, only a few of these enzymes have been characterized in the Gram-positive bacterium Bacillus subtilis, the workhorse industrial microorganism included in the Food and Drug Administration's GRAS (generally regarded as safe) list. In the present study, we report the identification of a novel 5'-nucleotidase gene from B. subtilis, yutF, which comprises 771 bp encoding a 256-amino-acid protein belonging to the IIA subfamily of the HADSF. The gene product is responsible for the major p-nitrophenyl phosphatase activity in B. subtilis. The yutF gene was overexpressed in Escherichia coli, and its product fused to a polyhistidine tag was purified and biochemically characterized as a soluble 5'-nucleotidase with broad substrate specificity. The recombinant YutF protein was found to hydrolyze various purine and pyrimidine 5'-nucleotides, showing preference for 5'-nucleoside monophosphates and, specifically, 5'-XMP. Recombinant YutF also exhibited phosphohydrolase activity toward nucleotide precursors, ribose-5-phosphate and 5-phosphoribosyl-1-pyrophosphate. Determination of the kinetic parameters of the enzyme revealed a low substrate specificity (Km values in the mM concentration range) and modest catalytic efficiencies with respect to substrates. An initial study of the regulation of yutF expression showed that the yutF gene is a component of the yutDEF transcription unit and that YutF overproduction positively influences yutDEF expression.

Dataset Information

Molecular characterization of a glucokinase with broad hexose specificity from Bacillus sphaericus strain C3-41.

Publications

Molecular characterization of a glucokinase with broad hexose specificity from Bacillus sphaericus strain C3-41.

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