Stereochemistry of the conversions of L-threonine and D-threonine into 2-oxobutanoate by the L-threonine and D-threonine dehydratases of Serratia marcescens.

Abstract

dl[3‐2H]Threonine, l‐[3‐2H]threonine, (2RS,3S)‐2‐amino[3‐2H1] butanoic acid, (2RS,3S) 2‐amino[3‐3H1]butanoic acid and dl‐2‐amino[3‐14C]butanoic acid were synthesised. l‐[3‐2‐H]Threonine was converted into l‐[4‐2H1]isoleucine by Serratia marcescens strain IHr313. (2RS,3S)‐2‐Amino[3‐2‐H1]butanoic acid was converted into l‐[4‐4‐H1]isoleucine by S. marcescens strain 149. Analysis by 220‐MHz NMR spectroscopy of the labelled l‐isoleucine produced showed that the same diastereotopic hydrogen at C‐4 was labelled in each experiment, proving that, during the conversion of l‐threonine into 2‐oxobutanoate mediated by biosynthetic l‐threonine dehydratase, the hydroxyl group at C‐3 was replaced by hydrogen with retention of configuration. S. marcescens strain 149 lacks l‐threonine dehydratase but possesses an inducible d‐threonine dehydratase. This strain converted dl‐[3‐2H]threonine into dl‐[4‐2H1]isoleucine with the deuterium located in the diastereotopic C‐4 hydrogen derived from the 3 pro‐R hydrogen of 2‐aminobutanoic acid. This result proved that during the conversion of d‐threonine into 2‐oxobutanoate mediated by d‐threonine dehydratase, the hydroxyl group at C‐3 is replaced by hydrogen with retention of configuration. These results also prove that the protonation at C‐3 of the proposed enamine intermediate in the transformations catalysed by threonine dehydratase is under enzymatic control. The present results, taken in conjunction with the independent assignment of the signals in the 220‐MHz NMR spectrum of l‐isoleucine due to the diastereotopic protons at C‐4, prove that during the ethyl migration step in l‐isoleucine biosynthesis, the configuration at the migrating centre is retained.