FUNCTIONAL-ANALYSIS OF ESCHERICHIA-COLI THREONINE DEHYDROGENASE BY MEANS OF MUTANT ISOLATION AND CHARACTERIZATION

The oxidation of L-threonine to 2-amino-3-ketobutyrate, as catalyzed b y L-threonine dehydrogenase, is the first step in the major pathway fo r threonine catabolism in both eukaryotes and prokaryotes. Threonine d ehydrogenase of E. coli has considerable amino-acid sequence homology with a number of Zn2+-containing, medium-chain alcohol dehydrogenases. In order to further explore structure/function interrelationships of E. coli threonine dehydrogenase, 35 alleles of tdh that imparted a no- growth or slow-growth phenotype on appropriate indicator media were is olated after mutagenesis with hydroxylamine. Within this collection, 1 4 mutants had single amino-acid changes that were divided into 4 group s: (a) amino-acid changes associated with proposed ligands to Zn2+; (b ) a substitution of one of several conserved glycine residues; (c) mut ations at the substrate or coenzyme binding site; (d) alterations that resulted in a change of charge near the active site. These findings u ncover previously unidentified amino-acid residues that are important for threonine dehydrogenase catalysis and also indicate that the three -dimensional structure of tetrameric E. coli threonine dehydrogenase h as considerable similarity with the dimeric horse liver alcohol dehydr ogenase.