Purification and properties of mycobacterial GDP-mannose pyrophosphorylase.
The enzyme that catalyzes the formation of GDP-d-mannose from GTP and alpha-d-mannose-1-P was purified about 2300-fold to near homogeneity from the soluble fraction of Mycobacterium smegmatis. At the final stage of purification, a major protein band of 37 kDa was observed and this band was specifically labeled, and in a concentration-dependent manner, by the photoaffinity probe 8-N3-GDP[32P]-d-mannose. The purified enzyme was stable for several months when kept in the frozen state. The 37-kDa band was subjected to protein sequencing and one peptide sequence of 25 amino acids showed over 80% identity to GDP-mannose pyrophosphorylases of pig liver and Saccharomyces cerevesiae. In contrast to some other bacterial GDP-mannose pyrophosphorylases, the mycobacterial enzyme was not multifunctional and did not have phosphomannose isomerase or phosphoglucose isomerase activity. Also, in contrast to the pig liver enzyme which uses mannose-1-P or glucose-1-P plus GTP to synthesize either GDP-mannose or GDP-glucose, the mycobacterial enzyme was specific for mannose-1-P as the sugar phosphate substrate. The enzyme was also relatively specific for GTP as the nucleoside triphosphate substrate. ITP was about 18% as effective as GTP, but ATP, CTP, and UTP were inactive. The activity of the enzyme was inhibited by GDP-glucose and glucose-1-P, although neither was a substrate for this enzyme. The pH optimum for the enzyme was 8.0, and Mg2+ was the best cation with optimum activity at about 5 mM. This enzyme is important for producing the activated form of mannose for formation of cell wall lipoarabinomannan and various mannose-containing glycolipids and polysaccharides.
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Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, 72205, USA.
Elbein
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D000595:Amino Acid Sequence; D000818:Animals; D004795:Enzyme Stability; D005958:Glucosephosphates / Q000494:pharmacology; D006155:Guanosine Diphosphate Mannose / Q000031:analogs & derivatives / Q000378:metabolism; D006156:Guanosine Diphosphate Sugars / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007293:Inosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008359:Mannose-6-Phosphate Isomerase / Q000378:metabolism; D008360:Mannosephosphates / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D020102:Mycobacterium smegmatis / Q000201:enzymology*; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019920:Photoaffinity Labels; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity