Occurrence of Transsulfuration in Synthesis ofl-Homocysteine in an Extremely Thermophilic Bacterium, Thermus thermophilus HB8
Occurrence of Transsulfuration in Synthesis ofl-Homocysteine in an Extremely Thermophilic Bacterium, Thermus thermophilus HB8
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极端嗜热细菌嗜热栖热菌 HB8 中 1-同型半胱氨酸合成中发生转硫作用
DOI：
10.1128/jb.183.6.2086-2092.2001
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发表时间：
2001
影响因子：
3.2
通讯作者：
T. Iwama
中科院分区：
文献类型：
--
作者：
S. Yamagata;Kazuhito Ichioka;K. Goto;Y. Mizuno;T. Iwama
在合成培养基中培养的嗜热热菌（Thermus thermophilus HB8）的细胞提取物，以o -乙酰-l-高丝氨酸和l-半胱氨酸为底物催化半胱硫氨酸γ-合成，但不以l-同型半胱氨酸和l-丝氨酸（oro -乙酰-l-丝氨酸）催化β-合成。通过测定细胞提取物中含硫氨基酸的催化活性，估计了合成酶代谢含硫氨基酸的数量。在培养基中添加l-蛋氨酸可显著抑制胱氨酸β-裂解酶（EC 4.4.1.8）和乙酰-l-丝氨酸巯基化酶（EC 4.2.99.8）的合成。0.5 mM的l-半胱氨酸和谷胱甘肽作为唯一硫源，分别抑制了55%和73%的o -乙酰丝氨酸巯基化酶的合成，证实了该酶作为半胱氨酸合成酶的功能。以同样的方式在1 ~ 5 mM处加入蛋氨酸，可使o -乙酰丝氨酸巯基水解酶的合成降低2.1 ~ 2.5倍。通过测定同型半胱氨酸在过量的o -乙酰丝氨酸存在下合成的半胱氨酸和巯基水解酶的纯化制剂，建立了一种测定同型半胱氨酸释放的低浓度硫化物（0.01 ~ 0.05 mM）的方法。细胞提取物对同型半胱氨酸γ-裂解酶反应有催化作用，比活性为5 ~ 7 nmol/min/mg蛋白质，对蛋氨酸γ-裂解酶反应无催化作用。这些结果表明，用蛋氨酸衍生的同型半胱氨酸中的硫在o -乙酰丝氨酸巯基化酶的催化条件下也可合成半胱氨酸。蛋氨酸明显抑制乙酰丝氨酸巯基化酶。在培养基中添加硫源对o -乙酰纯丝氨酸巯基化酶合成的影响以及蛋氨酸对该酶活性的抑制作用，大多通过假设生物体有两种具有o -乙酰纯丝氨酸巯基化酶活性的蛋白质来理解，其中一种是半胱硫氨酸γ-合成酶。虽然在遗传学研究的基础上已经报道了嗜热T. HB27通过o -乙酰丝氨酸巯基化酶的催化直接合成同型半胱氨酸(T. Kosuge， D. Gao，和T. Hoshino， J. Biosci。本研究对相关酶行为的研究结果表明，硫首先进入半胱氨酸，然后通过半胱甘氨酸inT转移到同型半胱氨酸。酸奶有助于HB8。
ABSTRACT A cell extract of an extremely thermophilic bacterium,Thermus thermophilus HB8, cultured in a synthetic medium catalyzed cystathionine γ-synthesis withO-acetyl-l-homoserine andl-cysteine as substrates but not β-synthesis withdl-homocysteine and l-serine (orO-acetyl-l-serine). The amounts of synthesized enzymes metabolizing sulfur-containing amino acids were estimated by determining their catalytic activities in cell extracts. The syntheses of cysthathionine β-lyase (EC 4.4.1.8 ) andO-acetyl-l-serine sulfhydrylase (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 medium as the sole sulfur source repressed the synthesis ofO-acetylserine sulfhydrylase by 55 and 73%, respectively, confirming that this enzyme functions 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) liberated from homocysteine by determining cysteine synthesized with it in the presence of excess amounts of O-acetylserine and a purified preparation of the sulfhydrylase was established. The extract of cells catalyzed the homocysteine γ-lyase reaction, with a specific activity of 5 to 7 nmol/min/mg of protein, but not the methionine γ-lyase reaction. These results suggested that cysteine was also synthesized under the conditions employed by the catalysis ofO-acetylserine sulfhydrylase using sulfur of homocysteine derived from methionine. Methionine inhibitedO-acetylserine sulfhydrylase markedly. The effects of sulfur sources added to the medium on the synthesis ofO-acetylhomoserine sulfhydrylase and the inhibition of the enzyme activity by methionine were mostly understood by assuming that the organism has two proteins having O-acetylhomoserine sulfhydrylase activity, one of which is cystathionine γ-synthase. Although it has been reported that homocysteine is directly synthesized in T. thermophilus HB27 by the catalysis ofO-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 indicate that sulfur is first incorporated into cysteine and then transferred to homocysteine via cystathionine inT. thermophilus HB8.