1. Physiological comparison of D-cysteine desulfhydrase of Escherichia coli with 3-chloro-D-alanine dehydrochlorinase of Pseudomonas putida CR 1-1
T Nagasawa, T Ishii, H Yamada Arch Microbiol. 1988;149(5):413-6. doi: 10.1007/BF00425580.
D-Cysteine desulfhydrase of Escherichia coli W3110 delta trpED102/F' delta trpED102 was physiologically characterized. It was found to be located in the cytosolic fraction, as 3-chloro-D-alanine dehydrochlorinase is. D-Cysteine desulfhydrase catalyzed not only the alpha, beta-elimination reaction of O-acetyl-D-serine to form pyruvate, acetic acid and ammonia, but also the beta-replacement reaction of O-acetyl-D-serine with sulfide to form D-cysteine. However, these reactions appeared not to proceed in vivo. No other activity of D-cysteine synthesis from O-acetyl-D-serine and sulfide was detected in a crude cell extract of E. coli which was immunotitrated with antibodies raised against the purified D-cysteine desulfhydrase. Although D-cysteine desulfhydrase catalyzes the degradation (alpha, beta-elimination reaction) of 3-chloro-D-alanine, which is an effective antibacterial agent, E. coli W3110 delta trpED102/F' delta trpED102 did not show resistance against 3-chloro-D-alanine. Therefore, D-cysteine desulfhydrase does not contribute to 3-chloro-D-alanine detoxification in vivo.
2. 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
T Nagasawa, H Ohkishi, B Kawakami, H Yamano, H Hosono, Y Tani, H Yamada J Biol Chem. 1982 Nov 25;257(22):13749-56.
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.
3. Inhibition of bacterial growth by beta-chloro-D-alanine
J M Manning, N E Merrifield, W M Jones, E C Gotschlich Proc Natl Acad Sci U S A. 1974 Feb;71(2):417-21. doi: 10.1073/pnas.71.2.417.
The D- and L-isomers of beta-chloroalanine inhibit the growth of Diplococcus pneumoniae, Streptococcus pyogenes, Bacillus subtilis, and Escherichia coli. With pneumococcus the inhibition by beta-chloro-D-alanine is completely prevented by either D-alanine or D-alanyl-D-alanine, while L-alanine is not effective in preventing the inhibition. The inhibition of growth by beta-chloro-L-alanine is not affected by D-alanine and is only partially prevented by high concentrations of L-alanine. The intracellular free alanine in untreated E. coli and B. subtilis is about 95% in the D-configuration while the free intracellular alanine in both organisms after treatment with beta-chloro-D-alanine is predominantly the L-isomer. These results suggested that the beta-chloroamino acid inactivates alanine racemase (EC 5.1.1.1). Indeed, when extracts of E. coli or B. subtilis were treated with beta-chloro-D-alanine, the activities of alanine racemase and of D-glutamate-D-alanine transaminase were found to be 90-95% inhibited. Studies with mice have shown that beta-chloro-D-alanine is an effective antibacterial agent in vivo againt D. pneumoniae, S. pyogenes, and E. coli.
