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J Bacteriol. 1981 October; 148(1): 72-82
Copyright © 1981, American Society for Microbiology. All Rights Reserved.

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

Daniel A. Kunz, Douglas W. Ribbons^1, and Peter J. Chapman²

¹ Department of Biochemistry, School of Medicine, University of Miami, Miami, Florida 33101,
² Department of Biochemistry, University of Minnesota, St. Paul, Minnesota 55108

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.

Present address: Central Research and Development Department, E. I. du Pont de Nemours & Co., Inc., Wilmington, DE 19898.

Present address: Department of Biochemistry and Soil Science, University College of North Wales, Bangor, Gwynedd LL57 2UW, Wales, United Kingdom.