Ketopantoic acid reductase of Pseudomonas maltophilia 845. Purification, characterization, and role in pantothenate biosynthesis.
Ketopantoic acid reductase (EC 1.1.1.169), an enzyme that catalyzes the formation of D-(-)-pantoic acid from ketopantoic acid, was purified 6,000-fold to apparent homogeneity with a 35% overall recovery from Pseudomonas maltophilia 845 and then crystallized. The relative molecular mass of the native enzyme, as estimated by the sedimentation equilibrium method, is 87,000 +/- 5,000, and the subunit molecular mass is 30,500. The enzyme shows high specificity for ketopantoic acid as a substrate (Km = 400 microM, Vm = 1,310 units/mg of protein) and NADPH as a coenzyme (Km = 31.8 microM). Only 2-keto-3-hydroxyisovalerate (Km = 8.55 mM, Vm = 35.8 units/mg) was reduced among a variety of other carbonyl compounds tested. The reaction is reversible (Km for D-(-)-pantoic acid = 52.1 mM), although the reaction equilibrium greatly favors the direction of D-(-)-pantoic acid formation. That the enzyme is responsible for the synthesis of D-(-)-pantoic acid necessary for the biosynthesis of pantothenic acid in P. maltophilia 845 is indicated by the observations that only this enzyme is missing in D-(-)-pantoate (or pantothenate)-requiring mutants derived from P. maltophilia 845 among several enzymes (i.e. ketopantoyl lactone reductase (EC 1.1.1.168) and acetohydroxy acid isomeroreductase (EC 1.1.1.86], which may be concerned in the formation of D-(-)-pantoic acid, assayed, whereas it is present in substantial amounts in the parent strain and in spontaneous revertants of the mutants.
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D002087:Butyrates / Q000378:metabolism; D002384:Catalysis; D002845:Chromatography; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D006885:Hydroxybutyrates / Q000096:biosynthesis; D007651:Keto Acids / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009154:Mutation; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010205:Pantothenic Acid / Q000096:biosynthesis*; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D013053:Spectrophotometry; D013379:Substrate Specificity
D000596:Amino Acids; D002087:Butyrates; D006885:Hydroxybutyrates; D007651:Keto Acids; C016031:pantoic acid; D010205:Pantothenic Acid; D009249:NADP; D000429:Alcohol Oxidoreductases; C021934:2-dehydropantoate 2-reductase