Stereospecificity of Pseudomonas fluorescens kynureninase for diastereomers of beta-methylkynurenine

The diastereomers of beta-methyl-L-kynurenine were prepared by preparative ozonolysis of the respective diastereomers of beta-methyl-L-tryptophan. A p ractical method for preparative enzymatic resolution of the diastereomers o f beta-methyltryptophan was developed using carboxypeptidase A digestion of the N-trifluoroacetyl derivatives. The stereochemical assignment was confi rmed by X-ray crystal structure determination of (2S,3R)-threo-beta-methyl- L-tryptophan. (2S,3S)-erythro-beta-Methyl-L-kynurenine is a slow substrate for kynureninase from Pseudomonas fluorescens (k(cat)/K-m=0.1% that of L-ky nurenine), producing anthranilic acid, while (2S,3R)-threo-L-kynurenine is about 390-fold less reactive than erythro. Rapid-scanning stopped-flow meas urements show that beta-methyl substitution affects the rate of alpha-depro tonation of the L-kynurenine-pyridoxal-5'-phosphate Schiffs base. This is c onsistent with the stereoelectronic requirements of the reaction. These res ults are the first demonstration that beta-substituted kynurenines can be s ubstrates for kynureninase, and may be useful in the design of mechanism-ba sed inhibitors. (C) 1999 Elsevier Science Ltd. All rights reserved.