Common Enzymes of Branched-Chain Amino Acid Catabolism in Pseudomonas putida

Abstract

Two types of Pseudomonas putida PpG2 mutants which were unable to degrade branched-chain amino acids were isolated after mutagenesis and selection for ability to grow on succinate, but not valine, as a sole source of carbon. These isolates were characterized by growth on the three branched-chain amino acids (valine, isoleucine, and leucine), on the corresponding branchedchain keto acids (2-ketoisovalerate, 2-keto-3-methylvalerate, and 2-ketoisocaproate), and on other selected intermediates as carbon sources, and by their enzymatic composition. One group of mutants lost 2-ketoisovalerate-inducible branched-chain keto acid dehydrogenase that was active on all three keto acids. There was also a concomitant loss of ability to grow on all three branched-chain amino acids as well as on all three corresponding keto acids, but there was retention of ability to use subsequent intermediates in the catabolism of branched-chain amino acids. Another type of mutant showed a marked reduction in branched-chain amino acid transaminase activity and grew poorly at the expense of all three amino acids, but it utilized subsequent intermediates as carbon sources. Both the transaminase and branched-chain keto acid dehydrogenase mutants retained the ability to degrade camphor. These findings are consistent with the view that branched-chain amino acid transaminase and branched-chain keto acid dehydrogenase are common enzymes in the catabolism of valine, isoleucine, and leucine.

d - and l -Isoleucine Metabolism and Regulation of Their Pathways in Pseudomonas putida

“…Induction of isoleucine catabolic enzymes. The data in Tables 2 and 3 are in agreement with earlier observations that two enzymes of the common pathway, D-amino acid dehydrogenase and branched-chain keto acid dehydrogenase, were induced by growth with any of the three branched-chain amino acids and that branched-chain keto acid dehydrogenase, but not D-amino acid dehydrogenase, was induced by growth in media with 2-keto-3-methylvalerate (14,16). Branched-chain amino acid transaminase is a constitutive enzyme (14).…”

“…The role of this enzyme in the cell must be an interesting one if it functions in both catabolism and biosynthesis of the branched-chain amino acids. It would be interesting to determine how the cell distinguishes between keto acids derived from catabolic pathways, which cause induction of branched-chain keto acid dehydrogenase (16), and keto acids derived from biosynthetic pathways, which do not induce branchedchain keto acid dehydrogenase. At least one and possibly two new enzymes were characterized during the course of this study, tiglyl-CoA hydrase and 2-methyl-3hydroxybutyryl-CoA dehydrogenase.…”

“…), oxidative decarboxylation (reaction II), two oxidative reactions (reactions III and V), and finally cleavage to acetyl-coenzyme A (CoA) and propionyl-CoA (reaction VI). Reactions I.A., I.B., and II are referred to as the common pathway (14,16) branched-chain D-amino acids (I.B. ), and branched-chain keto acid dehydrogenase is responsible for oxidation of branched-chain keto acids (II).…”

“…of Oklahoma Health Sciences Center, Oklahoma City, 1970). Martin et al (16) found that a mutant of P. putida unable to grow with any of the branched-chain amino acids as sole sources of carbon has lost the ability to oxidize the keto acids derived from valine, leucine, and isoleucine. Very little is known about reaction III catalyzed by branched-chain acyl-CoA dehydrogenase, although a similar activity was reported by Marshall and Sokatch in their study of valine catabolism in P. putida (14).…”

Purification of a branched-chain keto acid dehydrogenase from Pseudomonas putida

“…Our purified enzyme catalyzed the oxidation of 2ketoisovalerate, 2-ketoisocaproate, and 2-keto-3methylvalerate and is probably responsible for these reactions in intact cells during the metabolism of valine, leucine, and isoleucine. Support for this hypothesis comes from previous genetic studies which showed that mutants of P. putida that were unable to form active branched-chain keto acid dehydrogenase lacked the ability to oxidize these three keto acids but not pyruvate or 2-ketoglutarate (8). These mutants also lacked the ability to grow on valine, leucine, and isoleucine as sole carbon sources.…”