The 1.6 A crystal structure of Mycobacterium smegmatis MshC: the penultimate enzyme in the mycothiol biosynthetic pathway.
The 1.6 A crystal structure of Mycobacterium smegmatis MshC: the penultimate enzyme in the mycothiol biosynthetic pathway.
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DOI：
10.1021/bi801708f
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发表时间：
2008-12-16
期刊：
影响因子：
2.9
通讯作者：
Blanchard, J. S.
中科院分区：
文献类型：
--
作者：
Tremblay, L. W.;Fan, F.;Vetting, M. W.;Blanchard, J. S.
耻垢分枝杆菌 MshC 催化 GlcN-Ins 和 L-半胱氨酸的 ATP 依赖性缩合形成 L-Cys-GlcN-Ins，这是菌硫醇生物合成的倒数第二步。结晶天然全长 MshC 的尝试尚未成功。然而，将该酶与半胱氨酰腺苷酸类似物 5'-O-[N-(L-半胱氨酰)-氨磺酰]腺苷 (CSA) 一起孵育，然后进行 24 小时有限的胰蛋白酶蛋白水解，产生易于结晶的酶制剂。解出了活性位点结合有 CSA 的 MshC 的三维结构，并将其细化至 1.6 Å。精细结构表现出对应于整个 47 kD MshC 分子的电子密度，但 KMSKS 环（残基 285-297）除外，该环先前与相关 tRNA 合酶中腺苷酸的形成有关。 MshC 的整体三级折叠与半胱氨酰-tRNA 合成酶的三级折叠相似，具有罗斯曼折叠催化结构域。 CSA 硫醇盐与活性位点底部锌离子的相互作用表明金属离子参与氨基酸结合和区分。观察到许多活性位点残基与配体相互作用，表明在底物结合和催化中发挥作用。利用蛋白水解环建模和 GlcN-Ins 对接进行的分析，以及活性位点序列保守性的检查表明，半胱氨酰-tRNA 合成酶和 MshC 在识别各自反应的底物方面存在相似之处和差异。
Mycobacterium smegmatis MshC catalyzes the ATP-dependent condensation of GlcN-Ins and L-cysteine to form L-Cys-GlcN-Ins, the penultimate step in mycothiol biosynthesis. Attempts to crystallize the native, full-length MshC have been unsuccessful. However, incubation of the enzyme with the cysteinyl adenylate analogue, 5′-O-[N-(L-cysteinyl)-sulfamonyl]adenosine (CSA), followed by a 24-hour limited trypsin proteolysis yielded an enzyme preparation that readily crystallized. The three-dimensional structure of MshC with CSA bound in the active site was solved and refined to 1.6 Å. The refined structure exhibited electron density corresponding to the entire 47 kDalton MshC molecule, with the exception of the KMSKS loop (residues 285–297), a loop previously implicated in the formation of the adenylate in related tRNA synthases. The overall tertiary fold of MshC is similar to that of cysteinyl-tRNA synthetase, with a Rossmann fold catalytic domain. The interaction of the thiolate of CSA with a zinc ion at the base of the active site suggests that the metal ion participates in amino acid binding and discrimination. A number of active site residues were observed to interact with the ligand, suggesting a role in substrate binding and catalysis. Analysis utilizing modeling of the proteolyzed loop and GlcN-Ins docking, as well as the examination of sequence conservation in the active site suggests similarities and differences between cysteinyl-tRNA synthetases and MshC in recognition of the substrates for their respective reactions.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
11.4
作者：
Cusack, S;Yaremchuk, A;Tukalo, M
通讯作者：
Tukalo, M
影响因子：
11.4
作者：
Cavarelli, J;Delagoutte, B;Moras, D
通讯作者：
Moras, D
影响因子：
4.1
作者：
KIM, YS;KANG, SW
通讯作者：
KANG, SW
影响因子：
5.6
作者：
Kuratani, M;Sakai, H;Yokoyama, S
通讯作者：
Yokoyama, S