3-Chloro-D-alanine chloride-lyase (deaminating) of Pseudomonas putida CR 1.1. Purification and characterization of a novel enzyme occurring in 3-chloro-D-alanine-resistant pseudomonads.
A novel enzyme catalyzing cleavage of 3-chloro-D-alanine to pyruvate, ammonia, and chloride ion is distributed in some pseudomonads which have a resistance to high concentrations of 3-chloro-D-alanine. Pseudomonas putida CR 1-1 (AKU 867) was found to have the highest activity of enzyme, which was inducibly formed by the addition of 3-chloro-D-alanine to the medium. The enzyme, tentatively called 3-chloro-D-alanine chloride-lyase, was purified from P.l putida CR 1-1 in seven steps. After the last step, the enzyme appeared to be homogeneous by the criteria of polyacrylamide gel electrophoresis, analytical ultracentrifuge, and double diffusion in agarose. The enzyme has a molecular weight of about 76,000 and consists of two subunits identical in molecular weight (approximately 38,000). The enzyme exhibits absorption maxima at 278 nm and 418 nm, which are independent of the pH (6.0-9.0), and contains 2 mol of pyridoxal 5'-phosphate/mol of the enzyme. The holoenzyme is resolved to the apoenzyme by incubation with phenylhydrazine and reconstituted by the addition of pyridoxal-P. The apoenzyme can be crystallized by adding ammonium sulfate. 3-Chloro-D-alanine chloride-lyase catalyzes an alpha, beta-elimination reaction of 3-chloro-D-alanine and also, but to a lesser extent, D-cysteine and D-cysteine. The enzyme also catalyzes a beta-replacement reaction of chlorine of 3-chloro-D-alanine with hydrosulfide to yield D-cysteine. The important role of this novel beta-lyase enzyme in the detoxication of e-chloro-D-alanine by P. putida CR 1-1 is also discussed.
D000409:Alanine / Q000031:analogs & derivatives*; D001419:Bacteria / Q000201:enzymology; D003460:Crystallization; D004352:Drug Resistance, Microbial; D004790:Enzyme Induction; D007700:Kinetics; D008190:Lyases / Q000302:isolation & purification* / Q000378:metabolism; D046911:Macromolecular Substances; D008970:Molecular Weight; D011549:Pseudomonas / Q000201:enzymology*; D011732:Pyridoxal Phosphate / Q000032:analysis; D013045:Species Specificity; D015091:beta-Alanine / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000378:metabolism
D046911:Macromolecular Substances; D015091:beta-Alanine; C006977:3-chloroalanine; D011732:Pyridoxal Phosphate; D008190:Lyases; C031729:3-chloro-D-alanine dehydrochlorinase; D000409:Alanine