Stereospecificity of the Reductoisomerase-Catalyzed Step in the Pathway of Biosynthesis of Valine and Leucine

Abstract

Experiments are described which complete the determination of the stereochemistry of the intermediate steps in the biosynthesis of valine from pyruvate. Further, the preservation of stereospecificity on the pathway of biosynthesis of leucine from -ketoisovalerate has been examined. (RS)- [2-13CH3]Acetolactate (2-hydroxy-2- [13C]methyl-3-oxobutanoic acid) was added to cultures of Escherichia coli strain CU888 (blocked in the synthesis of acetolactate from pyruvate) on minimal medium supplemented with L-isoleucine. L-Valine isolated from the harvested cells was examined by carbon-13 nuclear magnetic resonance at 22.63 MHz and gave a single peak with a chemical shift of 17.8 ppm relative to tetramethylsilane. L-Leucine isolated from the same cells gave a single strong peak with a chemical shift of 21.4 ppm. These results establish the configuration of the isolated valine to be (2S,3S)-[4-13C]valine, identical with the synthetic product [Kluender, H., Bradley, C. H., Sih, C. J., Fawcett, P., & Abraham, E. P. (1973) J. Am. Chem. Soc. 95, 6149-6150], The stereochemistry of the reductoisomerase-catalyzed step is shown to be the conversion of (2S)-[2-13CH3]acetolactate to (2R,3R)-2,3-dihydroxy-3-[13C]methylbutanoic acid [cf. Armstrong, F. B., Hedgecock, C. J. R., Reary, J. B., Whitehouse, D., & Grout, D. H. G. (1974) J. Chem. Soc., Chem. Commun., 351-352, and Hill, R. K., & Yan, S-J. (1971) Bioorg. Chem. I, 446-456], The bioconversion of L-valine to L-leucine is proposed to occur without change in configuration of the terminal chiral center, yielding (2S,4S)-[5-13C]leucine. The results suggest methods for the preparation of L-valine and L-leucine isotopically labeled in specific positions. © 1979, American Chemical Society. All rights reserved.