Metabolism of Allylglycine and cis -Crotylglycine by Pseudomonas putida ( arvilla ) mt-2 Harboring a TOL Plasmid

Abstract

Spontaneous mutants which acquired the ability to utilize d -allylglycine ( d -2-amino-4-pentenoic acid) and dl - cis -crotylglycine ( dl -2-amino- cis -4-hexenoic acid) but not l -allylglycine or dl - trans -crotylglycine could be readily isolated from Pseudomonas putida mt-2 (PaM1). Derivative strains of PaM1 putatively cured of the TOL (pWWO) plasmid were incapable of forming mutants able to utilize the amino acids for growth; however, this ability could be regained by conjugative transfer of the TOL (pWWO) plasmid from a wild-type strain of mt-2 or of the TOL (pDK1) plasmid from a related strain of P. putida (HS1), into cured recipients. dl -Allylglycine-grown cells of one spontaneous mutant (PaM1000) extensively oxidized dl -allylglycine and dl - cis -crotylglycine, whereas only a limited oxidation was observed toward l -allylglycine and dl - trans -crotylglycine. Cell extracts prepared from PaM1000 cells contained high levels of 2-keto-4-hydroxyvalerate aldolase and 2-keto-4-pentenoic acid hydratase, the latter enzyme showing higher activity toward 2-keto- cis -4-hexenoic acid than toward the trans isomer. Levels of other enzymes of the TOL degradative pathway, including toluate oxidase, catechol-2,3-oxygenase, 2-hydroxymuconic semialdehyde hydrolase, and 2-hydroxymuconic semialdehyde dehydrogenase, were also found to be elevated after growth on allylglycine. Whole cells of a putative cured strain, PaM3, accumulated 2-keto-4-pentenoic acid from d -allylglycine, which was shown to be rapidly degraded by cell extracts of PaM1000 grown on dl -allylglycine. These same cell extracts were also capable of catalyzing the dehydrogenation of d - but not l -allylglycine and were further found to metabolize the amino acid completely to pyruvate and acetaldehyde. Differential centrifugation of crude cell extracts localized d -allylglycine dehydrogenase activity to membrane fractions. The results are consistent with a catabolic pathway for d -allylglycine and dl - cis -crotylglycine involving the corresponding keto-enoic acids as intermediates, the further metabolism of which is effected by the action of TOL plasmid-encoded enzymes.