Mechanistic insight into 3-deoxy-D-manno-octulosonate-8-phosphate synthase and 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase utilizing phosphorylated monosaccharide analogues.
Escherichia coli 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8-P) synthase is able to utilize the five-carbon phosphorylated monosaccharide, 2-deoxyribose 5-phosphate (2dR5P), as an alternate substrate, but not D-ribose 5-phosphate (R5P) nor the four carbon analogue D-erythrose 4-phosphate (E4P). However, E. coli KDO8-P synthase in the presence of either R5P or E4P catalyzes the rapid consumption of approximately 1 mol of PEP per active site, after which consumption of PEP slows to a negligible but measurable rate. The mechanism of this abortive utilization of PEP was investigated using [2,3-(13)C(2)]-PEP and [3-F]-PEP, and the reaction products were determined by (13)C, (31)P, and (19)F NMR to be pyruvate, phosphate, and 2-phosphoglyceric acid (2-PGA). The formation of pyruvate and 2-PGA suggests that the reaction catalyzed by KDO8-P synthase may be initiated via a nucleophilic attack to PEP by a water molecule. In experiments in which the homologous enzyme, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7-P) synthase was incubated with D,L-glyceraldehyde 3-phosphate (G3P) and [2,3-(13)C(2)]-PEP, pyruvate and phosphate were the predominant species formed, suggesting that the reaction catalyzed by DAH7-P synthase starts with a nucleophilic attack by water onto PEP as observed in E. coli KDO8-P synthase.
Howe
David L
DL
Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109-1065, USA.
Sundaram
Appavu K
AK
Wu
Jing
J
Gatti
Domenico L
DL
Woodard
Ronald W
RW
D010723:3-Deoxy-7-Phosphoheptulonate Synthase; D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002236:Carbohydrate Conformation; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009005:Monosaccharides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D009005:Monosaccharides; D011994:Recombinant Proteins; D010723:3-Deoxy-7-Phosphoheptulonate Synthase; C020011:2-dehydro-3-deoxyphosphooctonate aldolase; D000446:Aldehyde-Lyases