Functional analysis of E. coli threonine dehydrogenase by means of mutant isolation and characterization.
The oxidation of L-threonine to 2-amino-ketobutyrate, as catalyzed by L-threonine dehydrogenase, is the first step in the major pathway for threonine catabolism in both eukaryotes and prokaryotes. Threonine dehydrogenase of E. coli has considerable amino-acid sequence homology with a number of Zn(2+)-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 isolated after mutagenesis with hydroxylamine. Within this collection, 14 mutants had single amino-acid changes that were divided into 4 groups: (a) amino-acid changes associated with proposed ligands to Zn2+; (b) a substitution of one of several conserved glycine residues; (c) mutations at the substrate or coenzyme binding site; (d) alterations that resulted in a change of charge near the active site. These findings uncover 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 has considerable similarity with the dimeric horse liver alcohol dehydrogenase.
Chen
Y W
YW
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606, USA.
Dekker
E E
EE
Somerville
R L
RL
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013912:Threonine / Q000378:metabolism
D006898:Hydroxylamines; D019811:Hydroxylamine; D013912:Threonine; D000429:Alcohol Oxidoreductases; C023159:L-threonine 3-dehydrogenase