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Journal of Biochemistry Advance Access originally published online on July 29, 2008
Journal of Biochemistry 2008 144(4):507-512; doi:10.1093/jb/mvn094
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© 2008 The Japanese Biochemical Society.

Cloning and Characterization of Phosphoglucomutase and Phosphomannomutase Derived from Sphingomonas chungbukensis DJ77

Sung-Sook Yoon1, Sung-Ha Park1, Young-Chang Kim1, Malshik Shin2, Chom-Kyu Chong3 and Jung-Do Choi1,*

1School of Life Sciences and Biotechnology Research Institute, Chungbuk National University, Cheongju 371-763; 2Department of Food and Nutrition and Biofood Research Center, Chonnam National University, Gwangju 500-757; and 3Human Science Laboratory, Bioland Ltd, Ochang, Cheongwon, Chungbuk 363-883, Korea

*To whom correspondence should be addressed. Tel: +82-43-261-2308, Fax: +82-43-267-2306, E-mail: jdchoi{at}chungbuk.ac.kr

Received June 10, 2008; Accepted July 14, 2008


  Abstract

The enzymes phosphoglucomutase (PGM) and phosphomannomutase (PMM) play an important role in the synthesis of extracellular polysaccharide. By colony hybridization of the fosmid library of Sphingomonas chungbukensis DJ77, an open reading frame (ORF-1) of 1,626 nucleotides, whose predicted product is highly homologous with other PGM proteins from several bacterial species, was identified. An additional open reading frame (ORF-2) of 1,437 nucleotides was identified, and its encoded protein shows a high level of similarity with the PGM/PMM protein family. The two genes were cloned into a bacterial expression vector pET-15b (+) and expressed in Escherichia coli as fusion proteins with (His)6-tag. Both recombinant proteins (designated as SP-1 and SP-2 for ORF-1 and ORF-2, respectively) exhibited PGM and PMM activities. The molecular masses of subunits of SP-1 and SP-2 were estimated to be around 58 and 51 kDa from SDS–PAGE, respectively. However, molecular masses of SP-1 and SP-2 in their native condition were determined to be approximately 59.5 and 105.4 kDa, according to non-denaturing PAGE, respectively. The SP-1 protein has a preference for glucose-1-phosphate rather than mannose-1-phosphate, while the preferred substrate of SP-2 is mannose-1-phosphate. Thus, the existence of two proteins with bifunctional PGM/PMM activities was first found S. chungbukensis DJ77.

Key Words: extracellular polysaccharide, gene cloning, phosphoglucomutase, phosphomannomutase, Sphingomonas chungbukensis DJ77, synthesis


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