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Applied and Environmental Microbiology, July 2003, p. 3849-3857, Vol. 69, No. 7
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.7.3849-3857.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Characterization and Molecular Cloning of a Novel Enzyme, Inorganic Polyphosphate/ATP-Glucomannokinase, of Arthrobacter sp. Strain KM

Takako Mukai,1 Shigeyuki Kawai,1 Hirokazu Matsukawa,2 Yuhsi Matuo,2 and Kousaku Murata1*

Department of Basic and Applied Molecular Biotechnology, Division of Food and Biological Science, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011,1 Oriental Yeast Co., Ltd., Itabashi-ku, Tokyo 174-8505, Japan2

Received 26 November 2002/ Accepted 1 April 2003

A bacterium exhibiting activities of several inorganic polyphosphate [poly(P)]- and ATP-dependent kinases, including glucokinase, NAD kinase, mannokinase, and fructokinase, was isolated, determined to belong to the genus Arthrobacter, and designated Arthrobacter sp. strain KM. Among the kinases, a novel enzyme responsible for the poly(P)- and ATP-dependent mannokinase activities was purified 2,200-fold to homogeneity from a cell extract of the bacterium. The purified enzyme was a monomer with a molecular mass of 30 kDa. This enzyme phosphorylated glucose and mannose with a high affinity for glucose, utilizing poly(P) as well as ATP, and was designated poly(P)/ATP-glucomannokinase. The Km values of the enzyme for glucose, mannose, ATP, and hexametaphosphate were determined to be 0.50, 15, 0.20, and 0.02 mM, respectively. The catalytic sites for poly(P)-dependent phosphorylation and ATP-dependent phosphorylation of the enzyme were found to be shared, and the poly(P)-utilizing mechanism of the enzyme was shown to be nonprocessive. The gene encoding the poly(P)/ATP-glucomannokinase was cloned from Arthrobacter sp. strain KM, and its nucleotide sequence was determined. This gene contained an open reading frame consisting of 804 bp coding for a putative polypeptide with a calculated molecular mass of 29,480 Da. The deduced amino acid sequence of the polypeptide exhibited homology to the amino acid sequences of the poly(P)/ATP-glucokinase of Mycobacterium tuberculosis H37Rv (level of homology, 45%), ATP-dependent glucokinases of Corynebacterium glutamicum (45%), Renibacterium salmoninarum (45%), and Bacillus subtilis (35%), and proteins of bacteria belonging to the order Actinomyces whose functions are not known. Alignment of these homologous proteins revealed seven conserved regions. The mannose and poly(P) binding sites of poly(P)/ATP-glucomannokinase are discussed.

* Corresponding author. Mailing address: Department of Basic and Applied Molecular Biotechnology, Division of Food and Biological Science, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan. Phone: 81-774-38-3766. Fax: 81-774-38-3767. E-mail: kmurata{at}kais.kyoto-u.ac.jp.

Applied and Environmental Microbiology, July 2003, p. 3849-3857, Vol. 69, No. 7
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.7.3849-3857.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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  • Lunin, V. V., Li, Y., Schrag, J. D., Iannuzzi, P., Cygler, M., Matte, A. (2004). Crystal Structures of Escherichia coli ATP-Dependent Glucokinase and Its Complex with Glucose. J. Bacteriol. 186: 6915-6927 [Abstract] [Full Text]  


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