1. 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
H Hosono, Y Tani, H Yamano, H Yamada, B Kawakami, T Nagasawa, H Ohkishi 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.
2. The effect of beta-chloro D-alanine and L-cycloserine on the serine, phosphorus and palmitic acid uptake and metabolism of Tetrahymena lipids
P Kovács Acta Biol Hung . 1999;50(4):363-73.
The serine palmitoyltransferase inhibitors beta-chloro-D-alanine and L-cycloserine resulted in the uptake and metabolism of 3H-serine, 3H-palmitic acid and 32P significant alterations in the unicellular Tetrahymena pyriformis GL as compared to the untreated cells. In contrast with the higher eukariotic cells, by these treatments - except 5 mM L-cycloserine - the ceramide formation were not inhibited in Tetrahymena. L-cycloserine inhibited the conversion of phosphatidylserine (PS) to phosphatidyl-ethanolamine (PE) by decarboxylation, and the conversion of PE to phosphatidylcoline (PC) by methylation. The shorter L-cycloserine treatments caused lower, and the longer treatments higher label in glycerophospholipids. beta-chloro-D-alanine resulted in the glycerophospholids higher lipid precursor incorporation both in the shorter and longer treatments. Presumably beta-chloro-D-alanine treatments inhibit the transaminase activity, and the higher concentration (5 and 10 mM) proved to be toxic for Tetrahymena. We found differences between the metabolism of serine and palmitic acid labeled lipids in the beta-chloro-D-alanine and L-cycloserine treated groups. This phenomenon is probably due to a difference in the uptake of phospholipid head group component serine and hydrophobic tail precursor palmitic acid: the incorporation of palmitic acid in Tetrahymena is extremely quick, on the other hand, the uptake of serine is slower, a clear time dependence was measured.
3. 3-Chloro-DL-alanine resistance by L-methionine-alpha-deamino-gamma-mercaptomethane-lyase activity
Yoshio Nakano, Mamiko Yoshimura, Toshihiko Koga, Haruka Fukamachi FEBS Lett . 2002 Jul 17;523(1-3):119-22. doi: 10.1016/s0014-5793(02)02958-7.
The antibacterial agent 3-chloro-DL-alanine (3CA) is an inhibitor of peptidoglycan synthesis. Fusobacterium nucleatum and Porphyromonas gingivalis, the bacteria responsible for oral malodor, are shown to be resistant to 1 mM 3CA, whereas Streptococcus mutans and Escherichia coli are sensitive to this antibacterial agent at the same concentration. We isolated the 3CA resistance gene from F. nucleatum and showed that the gene encodes an L-methionine-alpha-deamino-gamma-mercaptomethane-lyase that catalyzes the alpha,gamma-elimination of L-methionine to produce methyl mercaptan. The enzyme also exhibits 3CA chloride-lyase (deaminating) activity. This antibacterial agent is expected to be useful for specific selection of malodorous oral bacteria producing high amounts of methyl mercaptan.
