Identification of amino acid residues important for the phosphomannose isomerase activity of PslB in Pseudomonas aeruginosa PAO1.
Phosphomannose isomerase (PMI) plays a pivotal role in biosynthesis of GDP-mannose, an important precursor of many polysaccharides. We demonstrate in this study that Pseudomonas aeruginosa pslB encodes a protein with GDP-mannose pyrophosphorylase/PMI dual activities. The PMI activity is Co2+-dependent and could be inhibited by GDP-mannose in a competitive manner. Furthermore, the activity could be inactivated by 2,3-butanedione suggesting the presence of a catalytic Arg residue. Site-specific mutations at R373, R472, R479, E410, H411, N433 and E458 increase the KM approximately 8-20-fold. The PMI activity of PslB was completely diminished with a R408K or R408A, reflecting the importance of this residue in catalysis. Overall, these results provide a basis for understanding the catalytic mechanism of PMI.
Lee
Hui-Ju
HJ
College of Biological Science and Technology, National Chiao Tung University, 75 Po Ai Street, Hsin Chu, Taiwan.
Chang
Hwan-You
HY
Venkatesan
Nandinin
N
Peng
Hwei-Ling
HL
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D003035:Cobalt / Q000378:metabolism; D004252:DNA Mutational Analysis; D003931:Diacetyl / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism / Q000494:pharmacology; D008359:Mannose-6-Phosphate Isomerase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D006155:Guanosine Diphosphate Mannose; D003035:Cobalt; D001120:Arginine; D008359:Mannose-6-Phosphate Isomerase; D003931:Diacetyl