Occurrence of transsulfuration in synthesis of L-homocysteine in an extremely thermophilic bacterium, Thermus thermophilus HB8

A cell extract of an extremely thermophilic bacterium, Thermus thermophilus HB8, cultured in a synthetic medium catalyzed cystathionine gamma -synthes is with O-acetyl-L-homoserine and L-cysteine as substrates but not P-synthe sis with DL-homocysteine and L-serine (or O-acetyl-L-serine). The amounts o f synthesized enzymes metabolizing sulfur-containing amino acids were estim ated by determining their catalytic activities in cell extracts. The synthe ses of cysthathionine beta -lyase (EC 4.4.1.8) and O-acetyl-L-serine sulfhy drylase (EC 4.2.99.8) were markedly repressed by L-methionine supplemented to the medium, L-Cysteine and glutathione, both at 0.5 mM, added to the med ium as the sole sulfur source repressed the synthesis of O-acetylserine sul fhydrylase by 55 and 73%, respectively, confirming that this enzyme functio ns as a cysteine synthase, Methionine employed at 1 to 5 mM in the same way derepressed the synthesis of O-acetylserine sulfhydrylase 2.1- to 2.5-fold . A method for assaying a low concentration of sulfide (0.01 to 0.05 mM) li berated from homocysteine by determining cysteine synthesized with it in th e presence of excess amounts of O-acetylserine and a purified preparation o f the sulfhydrylase was established. The extract of tells catalyzed the hom ocysteine gamma -lyase reaction, with a specific activity of 5 to 7 nmol/mi n/mg of protein, but not the methionine gamma -lyase reaction. These result s suggested that cysteine was also synthesized under the conditions employe d by the catalysis of O-acetylserine sulfhydrylase using sulfur of homocyst eine derived from methionine. Methionine inhibited O-acetylserine sulfhydry lase markedly, The effects of sulfur sources added to the medium on the syn thesis of O-acetylhomoserine sulfhydrylase and the inhibition of the enzyme activity by methionine were mostly understood by assuming that the organis m has two proteins having O-acetylhomoserine sulfhydrylase activity, one of which is cystathionine gamma -synthase, Although it has been reported that homocysteine is directly synthesized in T. thermophilus HB27 by the cataly sis of O-acetylhomoserine sulfhydrylase on the basis of genetic studies (T, Kosuge, D, Gao, and T, Hoshino, J, Biosci, Bioeng, 90:271-279, 2000), the results obtained in this study for the behaviors of related enzymes indicat e that sulfur is first incorporated into cysteine and then transferred to h omocysteine via cystathionine in T, thermophilus HB8.