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

Spontaneous mutants which acquired the ability to utilized -allylglycine (d -2-amino-4-pentenoic acid) anddl -cis -crotylglycine (dl -2-amino-cis -4-hexenoic acid) but notl -allylglycine ordl -trans -crotylglycine could be readily isolated fromPseudomonas 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 ofP. putida (HS1), into cured recipients.dl -Allylglycine-grown cells of one spontaneous mutant (PaM1000) extensively oxidizeddl -allylglycine anddl -cis -crotylglycine, whereas only a limited oxidation was observed towardl -allylglycine anddl -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 thetrans 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 fromd -allylglycine, which was shown to be rapidly degraded by cell extracts of PaM1000 grown ondl -allylglycine. These same cell extracts were also capable of catalyzing the dehydrogenation ofd - but notl -allylglycine and were further found to metabolize the amino acid completely to pyruvate and acetaldehyde. Differential centrifugation of crude cell extracts localizedd -allylglycine dehydrogenase activity to membrane fractions. The results are consistent with a catabolic pathway ford -allylglycine anddl -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.