Stereospecificity of Pseudomonas fluorescens kynureninase for diastereomers of β-methylkynurenine Original Research Article

The diastereomers of β-methyl-l-kynurenine were prepared by preparative ozonolysis of the respective diastereomers of β-methyl-l-tryptophan. A practical method for preparative enzymatic resolution of the diastereomers of β-methyltryptophan was developed using carboxypeptidase A digestion of the N-trifluoroacetyl derivatives. The stereochemical assignment was confirmed by X-ray crystal structure determination of (2S,3R)-threo-β-methyl-l-tryptophan. (2S,3S)-erythro-β-Methyl-l-kynurenine is a slow substrate for kynureninase from Pseudomonas fluorescens (kcat/Km=0.1% that of l-kynurenine), producing anthranilic acid, while (2S,3R)-threo-l-kynurenine is about 390-fold less reactive than erythro. Rapid-scanning stopped-flow measurements show that β-methyl substitution affects the rate of α-deprotonation of the l-kynurenine-pyridoxal-5′-phosphate Schiffʹs base. This is consistent with the stereoelectronic requirements of the reaction. These results are the first demonstration that β-substituted kynurenines can be substrates for kynureninase, and may be useful in the design of mechanism-based inhibitors.