Biological Crystallography High-resolution Structure of the M14-type Cytosolic Carboxypeptidase from Burkholderia Cenocepacia Refined Exploiting Pdb_redo Strategies
Biological Crystallography High-resolution Structure of the M14-type Cytosolic Carboxypeptidase from Burkholderia Cenocepacia Refined Exploiting Pdb_redo Strategies
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新洋葱伯克霍尔德菌 M14 型胞质羧肽酶的生物晶体学高分辨率结构精炼利用 Pdb_redo 策略
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通讯作者：
W. Hunter
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作者：
V. Rimsa;T. Eadsforth;R. Joosten;W. Hunter
一个潜在的胞质β-羧肽酶从伯克霍尔德菌cenocepacia已结晶和同步辐射微焦点光束线允许收购衍射数据到1.9 A的分辨率。不对称单元包括含有超过1500个氨基酸的四聚体，并且嵌入PDB_REDO中的高通量自动化方案与模型图谱检查结合在一起进行改进。这种方法突出了这种协议对于有效分析的价值。该亚基由两个结构域构成。N-末端结构域以前仅在胞质羧肽酶（CCP）蛋白中观察到。C-末端结构域，其携带含Zn 2+的活性位点，用于将该蛋白质分类为羧肽酶的M14 D亚家族的成员。虽然真核CCP具有脱谷氨酰胺酶活性，并参与加工修饰的微管蛋白，但细菌家族成员的功能和底物仍然未知。B。Cenoce-Pacia蛋白对呋喃丙烯酰基谷氨酸衍生物（一种潜在的底物）没有表现出脱谷氨酰胺酶活性。先前显示的与二价阳离子配位的残基以及在相关酶如牛羧肽酶中有助于肽键断裂的残基是保守的。保守的基本补丁的位置在活性位点附近的催化Zn 2+，其中一个乙酸根离子被确定，表明识别的羧基末端以类似的方式，以其他羧肽酶。然而，有显着的差异，表明具有不同属性的基板的识别。值得注意的是在S10识别亚位点中存在赖氨酸，这表明对酸性底物具有特异性。
A potential cytosolic metallocarboxypeptidase from Burk-holderia cenocepacia has been crystallized and a synchrotron-radiation microfocus beamline allowed the acquisition of diffraction data to 1.9 A ˚ resolution. The asymmetric unit comprises a tetramer containing over 1500 amino acids, and the high-throughput automated protocols embedded in PDB_REDO were coupled with model–map inspections in refinement. This approach has highlighted the value of such protocols for efficient analyses. The subunit is constructed from two domains. The N-terminal domain has previously only been observed in cytosolic carboxypeptidase (CCP) proteins. The C-terminal domain, which carries the Zn 2+-containing active site, serves to classify this protein as a member of the M14D subfamily of carboxypeptidases. Although eukaryotic CCPs possess deglutamylase activity and are implicated in processing modified tubulin, the function and substrates of the bacterial family members remain unknown. The B. cenoce-pacia protein did not display deglutamylase activity towards a furylacryloyl glutamate derivative, a potential substrate. Residues previously shown to coordinate the divalent cation and that contribute to peptide-bond cleavage in related enzymes such as bovine carboxypeptidase are conserved. The location of a conserved basic patch in the active site adjacent to the catalytic Zn 2+ , where an acetate ion is identified, suggests recognition of the carboxy-terminus in a similar fashion to other carboxypeptidases. However, there are significant differences that indicate the recognition of substrates with different properties. Of note is the presence of a lysine in the S1 0 recognition subsite that suggests specificity towards an acidic substrate.