Ozaki H, Shiio I
J Biochem. 1982 Apr;91(4):1163-71. doi: 10.1093/oxfordjournals.jbchem.a133799.
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.
在27株黄色短杆菌甲硫氨酸营养缺陷型菌株中，14株在高丝氨酸上不能生长，但在O - 乙酰高丝氨酸上能生长，并且发现所有这些菌株仅缺乏高丝氨酸O - 乙酰转移酶[EC 2.3.1.31]。另外3株在O - 乙酰高丝氨酸上不能生长，但在同型半胱氨酸上能生长，并且所测试的两株菌株仅缺乏O - 乙酰高丝氨酸巯基酶（AHS），而胱硫醚γ - 合酶[EC4.2.99.9]和β - 胱硫醚酶[EC 4.4.1.8]的活性没有任何变化。缺乏AHS的突变体的原养型回复突变体显示AHS活性同时回复。没有一株甲硫氨酸营养缺陷型菌株能在胱硫醚上生长。从这些结果可以得出结论，该细菌的甲硫氨酸生物合成途径涉及通过高丝氨酸O - 乙酰转移酶的作用由高丝氨酸形成O - 乙酰高丝氨酸，以及通过AHS反应由O - 乙酰高丝氨酸直接形成同型半胱氨酸。甲硫氨酸强烈抑制AHS的合成。AHS被纯化至纯度为70%。纯化后的制剂在用羟胺处理后被磷酸吡哆醛激活。该酶的分子量为360,000，活性的最适pH为8.7，与O - 乙酰 - L - 高丝氨酸特异性反应，与O - 乙酰 - L - 丝氨酸反应的活性仅为与O - 乙酰 - L - 高丝氨酸反应活性的百分之一，但与O - 琥珀酰 - L - 高丝氨酸、高丝氨酸或丝氨酸不显示活性。O - 乙酰高丝氨酸和H2S的Km值分别为2.0 mM和0.08 mM。该酶分别被10 mM L - 甲硫氨酸、L - 高丝氨酸和O - 乙酰 - L - 丝氨酸抑制50%、23%和29%，但不被胱硫醚或S - 腺苷 - L - 甲硫氨酸抑制。
