Methionine biosynthesis in Brevibacterium flavum: properties and essential role of O-acetylhomoserine sulfhydrylase.
Out of 27 strains of methionine auxotrophs of Brevibacterium flavum, 14 strains did not grow on homoserine but grew on O-acetylhomoserine, and all were found to lack homoserine O-acetyltransferase [EC 2.3.1.31] alone. Another 3 strains did not grow on O-acetylhomoserine but grew on homocysteine, and the two strains tested were found to lack O-acetylhomoserine sulfhydrylase (AHS) alone, without any changes in the activities of cystathionine gamma-synthase [EC4.2.99.9] and beta-cystathionase [EC 4.4.1.8]. Prototrophic revertants of the AHS-lacking mutants showed concomitant reversion of AHS activity. None of the methionine auxotrophs grew on cystathionine. From these results it was concluded that the methionine biosynthetic pathway of this bacterium involves formation of O-acetylhomoserine from homoserine by the action of homoserine O-acetyltransferase, and direct formation of homocysteine from O-acetylhomoserine by the AHS reaction. AHS synthesis was strongly repressed by methionine. AHS was purified to 70% purity. The purified preparation was activated by pyridoxal phosphate after treatment with hydroxylamine. The enzyme showed a molecular weight of 360,000, an optimum pH of 8.7 for activity, and specifically reacted with O-acetyl-L-homoserine and showed with O-acetyl-L-serine one hundredth as much activity as that with O-acetylo -homoserine, but did not show activity with O-succinyl-L-homoserine, homoserine, or serine. The Km values for O-acetylhomoserine and H2S were 2.0 mM and 0.08 mM, respectively. The enzyme was inhibited 50, 23. and 29% by 10 mM L-methionine, l-homoserine, and O-acetyl-L-serine, respectively, but it was not inhibited by cystathionine or S-adenosyl-L-methionine.
D001951:Brevibacterium / Q000254:growth & development / Q000378:metabolism*; D019757:Carbon-Oxygen Lyases; D003547:Cysteine Synthase; D006710:Homocysteine / Q000096:biosynthesis; D007700:Kinetics; D008190:Lyases / Q000302:isolation & purification / Q000378:metabolism*; D008715:Methionine / Q000096:biosynthesis*; D009097:Multienzyme Complexes; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D029701:Saccharomyces cerevisiae Proteins; D006710:Homocysteine; D008715:Methionine; D003547:Cysteine Synthase; C103381:MET17 protein, S cerevisiae; C018725:O-acetylhomoserine (thiol)-lyase; D008190:Lyases; D019757:Carbon-Oxygen Lyases