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D- and L-Isoleucine Metabolism and Regulation of Their Pathways in Pseudomonas putida

^a Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190

ABSTRACT

Pseudomonas putida oxidized isoleucine to acetyl-coenzyme A (CoA) and propionyl-CoA by a pathway which involved deamination of D-isoleucine by oxidation and L-isoleucine by transamination, oxidative decarboxylation, and beta oxidation at the ethyl side chain. At least three separate inductive events were required to form all of the enzymes of the pathway: D-amino acid dehydrogenase was induced during growth in the presence of D-isoleucine; branched-chain keto dehydrogenase was induced during growth on 2-keto-3-methylvalerate and enzymes specific for isoleucine metabolism; tiglyl-CoA hydrase and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase were induced by growth on isoleucine, 2-keto-3-methylvalerate, 2-methylbutyrate, or tiglate. Tiglyl-CoA hydrase and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase were purified simultaneously by several enzyme concentration procedures, but were separated by isoelectric focusing. Isoelectric points, pH optima, substrate specificity, and requirements for enzyme action were determined for both enzymes. Evidence was obtained that the dehydrogenase catalyzed the oxidation of 2-methyl-3-hydroxybutyryl-CoA to 2-methylacetoacetyl-CoA. 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase catalyzed the oxidation of 3-hydroxybutyryl-CoA, but L-3-hydroxyacyl-CoA dehydrogenase from pig heart did not catalyze the oxidation of 2-methyl-3-hydroxybutyryl-CoA; therefore, they appeared to be different dehydrogenases. Furthermore, growth on tiglate resulted in the induction of tiglyl-CoA hydrase and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase, but these two enzymes were not induced during growth on crotonate or 3-hydroxybutyrate.

¹ Present address: Department of Microbiology, Baylor University College of Medicine, Houston, Tex. 77025.

² Present address: Oklahoma Medical Research Foundation, Oklahoma City, Okla. 73104.

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