Abstract
Cystathionine gamma-lyase (EC 4.4.1.1) is widely distributed in actinomycetes, e.g. genera Streptomyces, Micromonospora, Micropolyspora, Mycobacterium, Nocardia, Streptosporangium, and Streptoverticillium. The enzyme was purified from Streptomyces phaeochromogenes (IFO 3105) in nine steps. After the last steps, the enzyme appeared to be homogenous by the criteria of polyacrylamide gel electrophoresis, analytical centrifugation, and double diffusion in agarose. The enzyme crystallized in the apo form with the addition of ammonium sulfate. The enzyme has a molecular weight of about 166,000 and consists of four subunits identical in molecular weight. The enzyme exhibits absorption maxima at 278 and 421 nm and contains 4 mol of pyridoxal 5'-phosphate/mol of enzyme. L-Cystathionine, L-homoserine, DL-lanthionine, L-djenkolic acid, and L-cystine are cleaved as preferred substrates by the Streptomyces enzyme. The alpha, beta-elimination reaction of L-cystathionine is also catalyzed by the enzyme at a ratio of about one-seventh of the alpha, gamma-elimination reaction. Cystathionine beta-synthase (EC 4.2.1.22) and cystathionine gamma-synthase (EC 4.2.99.9) activities were also detected in crude extracts of S. phaeochromogenes, but cystathionine beta-lyase (EC 4.4.1.8) was not. Consequently, the reverse transsulfuration pathway in actinomycetes may be similar to that in yeast and molds.
Highlights
The enzyme was purified from Streptomyces phaeo- The sulfur atom of cephalosporin C is principally supplied by chromogenes (IF0 3105) in nine steps
Treichler et al [12, 13] have recently demonstrated that cephalosporin C formation depends on functional cystathionine y-lyase; mutants having impaired cystathionine y-lyase are impaired in @-lactamantibiotic formation
Fifty actinomycetous strains from stock cultures of our laboratory (AKU-type culture collection) and IFO-type culture collection were tested for their ability to form the enzyme which catalyzes the a,y-elimination reaction of L-cystathionine
Summary
The reaction was carried out at 30"C in cuvettes containing 2 ml of the following solution: 0.16 mmol of potassium phosphate buffer (pH 8.0),0.15 pmol of pyridoxal-P, 0.26 pmol of NADH, 10 units of pig heart lactate dehydrogenase, the substrate, and an appropriateamount of the enzyme. The assay was carried out at 30 "C for 30 min in a reaction mixture (0.5 ml) consisting of 168 pmol of potassium phosphate buffer (pH 8.0), 32 nmol of pyridoxal-P, 12.5 pmol of 0-succinyl-L-homoserine (or 0acetyl-L-homoserine), 5 pmol of L-cysteine, and an appropriate amount of antiserum-treated cell-free extract.
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