Abstract
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.
Highlights
Ketopantoicacidreductase (EC 1.1.1.169), an en- and ketopantoyl lactone reductase (EC 1.1.1.168),present in zyme thatcatalyzesthe formation of D-(-)-pantoic acida crude extract of Saccharomyces cerevisiae
When the cellular level of ketoenzyme is responsible for thsyenthesisof D-(-)-pantoic pantoic acid is raised or an elevated level of this enzyme is acid necessary for the biosyntheosifs pantothenic acid presentcan it sufficiently function for the biosynthesis of in P. mdtophilia845 isindicated by the observations pantothenic acid
They found that an additional MP-indethat only this enzyme is missing in D-(-)-pantoate (or pendent activity for ketopantoic acid reduction, which correpantothenatebrequiring mutants derived froPm. mal- sponds to the ketopantoic acid reductase reported by King tophilia 845 among several enzymes (i.e. ketopantoyl and Wilken [4],is presentin crude extracts of S. typhimurium
Summary
$ Supported by a Scientific Cooperation Program of the Japan Society for the Promotion of Science, National University of Singa-. Dihydroxy-3,3-dimethylbutyricacid and itsy-lactone, respectively, ethyl-2,3-furandion, and dihydro-4-methyl-4-propyl-2,3-furandion and ketopantoic acid and ketopantoyl lactone for 2-keto-4-hydroxy- were kind gifts from Seitetsu Kagaku Co., Osaka The potassium salts of D-(-)- and L-(+)pantoic acid were prepared from the corresponding enantiomers of pantoyl lactone by treatment with KOH [11].2-Acetolactate and 2keto-3-hydroxyisovaleratwe ere synthesized as described by Krampitz [12] and Radhakrishnan et al [13], respectively. Standard proteins for high performance gel permeation chromatography, sodium dodecyl sulfate (SDS)*-polyacrylamidegel electrophoresis, and analytical gel filtration on a Sephacryl S-300 column were purchased from Oriental Yeast Co., Pharmacia Fine Chemicals Co., Uppsala, and Boehringer Mannheim, respectively. All other chemicals used in this work were of analytical grade and commercially available
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
