Cloning and characterization of phosphoglucomutase and phosphomannomutase derived from Sphingomonas chungbukensis DJ77.
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.
Yoon
Sung-Sook
SS
School of Life Sciences and Biotechnology Research Institute, Chungbuk National University, Cheongju, Korea.
Park
Sung-Ha
SH
Kim
Young-Chang
YC
Shin
Malshik
M
Chong
Chom-Kyu
CK
Choi
Jung-Do
JD
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D016366:Open Reading Frames; D010733:Phosphoglucomutase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017875:Phosphotransferases (Phosphomutases) / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D020578:Sphingomonas / Q000201:enzymology* / Q000235:genetics*
D017931:DNA Primers; D004269:DNA, Bacterial; D017875:Phosphotransferases (Phosphomutases); D010733:Phosphoglucomutase; C052799:phosphomannomutase