Stereochemistry of the Conversions of l ‐Threonine and d ‐Threonine into 2‐Oxobutanoate by the l ‐Threonine and d ‐Threonine Dehydratases of Serratia marcescens

1 dl [3‐ 2 H]Threonine, l ‐[3‐ 2 H]threonine, (2RS,3S)‐2‐amino[3‐ 2 H 1 ] butanoic acid, ( 2RS,3S ) 2‐amino[3‐ 3 H 1 ]butanoic acid and dl ‐2‐amino[3‐ 14 C]butanoic acid were synthesised. 2 l ‐[3‐ 2 ‐H]Threonine was converted into l ‐[4‐ 2 H 1 ]isoleucine by Serratia marcescens strain IHr313. 3 (2 RS ,3 S )‐2‐Amino[3‐ 2 ‐H 1 ]butanoic acid was converted into l ‐[4‐ 4 ‐H 1 ]isoleucine by S. marcescens strain 149. 4 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. 5 S. marcescens strain 149 lacks l ‐threonine dehydratase but possesses an inducible d ‐threonine dehydratase. This strain converted dl ‐[3‐ 2 H]threonine into dl ‐[4‐ 2 H 1 ]isoleucine with the deuterium located in the diastereotopic C‐4 hydrogen derived from the 3 pro‐R hydrogen of 2‐aminobutanoic acid. 6 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. 7 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. 8 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.