Characterization of glutamate‐cysteine ligase and glutathione synthetase from the δ‐proteobacterium Myxococcus xanthus

Misaki Okada, Y. Kimura
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引用次数: 3

Abstract

Glutathione (GSH) is synthesized in two ATP‐dependent reactions by glutamate‐cysteine ligase (Gcl) and glutathione synthetase (Gs). Myxococcus xanthus, a gram‐negative bacterium belonging to δ‐proteobacteria, possesses mxGcl and mxGs, which have high sequence identity with the enzymes from plants and bacteria, respectively. MxGcl2 was activated by Mn2+, but not by Mg2+, and stabilized in the presence of 5 mM Mn2+ or Mg2+. Sequence comparison of mxGcl2 and Brassica juncea Gcl indicated that they have the same active site residues, except for Tyr330, which interacts with Cys and which in mxGcl2 is represented by Leu267. The substitution of Leu267 with Tyr resulted in the loss of mxGcl2 activity, but that with Met (found in cyanobacterial Gcls) increased the mxGcl2 affinity for Cys. GSH and its oxidized form GSSG equally inhibited the activity of mxGcl2; the inhibition was augmented by ATP at concentrations >3 mM. Buthionine sulfoximine inactivated mxGcl2 with Ki = 2.1 μM, which was lower than those for Gcls from other organisms. The mxGcl2 activity was also suppressed by pyrophosphate and polyphosphates. MxGs was a dimer, and its activity was induced by Mg2+ but strongly inhibited by Mn2+ even in the presence of 10 mM Mg2+. MxGs was inhibited by GSSG at Ki = 3.6 mM. Approximately 1 mM GSH was generated with 3 units of mxGcl2 and 6 units of mxGs from 5 mM Glu, Cys, and Gly, and 10 mM ATP. Our results suggest that GSH production in M. xanthus mostly depends on mxGcl2 activity.
黄原粘球菌δ -变形菌中谷氨酸-半胱氨酸连接酶和谷胱甘肽合成酶的研究
谷胱甘肽(GSH)是由谷氨酸-半胱氨酸连接酶(Gcl)和谷胱甘肽合成酶(Gs)两种ATP依赖性反应合成的。黄粘球菌(Myxococcus xanthus)是一种革兰氏阴性细菌,属于δ -变形菌门,具有mxGcl和mxGs,它们分别与植物和细菌中的酶具有高度的序列一致性。MxGcl2被Mn2+激活,而不被Mg2+激活,在5 mM Mn2+或Mg2+存在下稳定。mxGcl2与芥菜Gcl的序列比较表明,除了与Cys相互作用的Tyr330和在mxGcl2中以Leu267为代表的Tyr330外,它们具有相同的活性位点残基。用Tyr取代Leu267导致mxGcl2活性的丧失,而用Met(在蓝藻Gcls中发现)取代则增加了mxGcl2对Cys的亲和力。GSH及其氧化形式GSSG对mxGcl2活性的抑制作用相同;浓度为bbb30 mM的ATP增强了抑制作用。丁硫氨酸亚砜对mxGcl2的灭活作用Ki = 2.1 μM,低于其他生物的灭活作用。mxGcl2活性也受到焦磷酸盐和多磷酸盐的抑制。MxGs是一种二聚体,其活性受Mg2+的诱导,但在Mg2+浓度为10 mM时,其活性仍被Mn2+强烈抑制。在Ki = 3.6 mM时,GSSG对MxGs有抑制作用。5 mM Glu、Cys和Gly产生3单位mxGcl2和6单位mxGs,以及10 mM ATP,产生约1 mM GSH。我们的研究结果表明,黄原草中GSH的产生主要取决于mxGcl2的活性。
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