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首页> 外文期刊>Biochemistry >Divergence of Structure and Function in the Haloacid Dehalogenase Enzyme Superfamily: Bacteroides thetaiotaomicron BT2127 Is an Inorganic Pyrophosphatase
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Divergence of Structure and Function in the Haloacid Dehalogenase Enzyme Superfamily: Bacteroides thetaiotaomicron BT2127 Is an Inorganic Pyrophosphatase

机译：卤素脱卤酶酶超家族的结构和功能的分歧：拟菌αOmicronBT2127是一种无机焦磷酸酶

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摘要

The explosion of protein sequence information requires that current strategies for function assignment evolve to complement experimental approaches with computationally based function prediction. This necessitates the development of strategies based on the identification of sequence markers in the form of specificity determinants and a more informed definition of orthologues. Herein, we have undertaken the function assignment of the unknown haloalkanoate dehalogenase superfamily member BT2127 (Uniprot accession code Q8A5 V9) from Bacteroides thetaiotaomicron using an integrated bioinformatics-structure-mechanism approach. The substrate specificity profile and steady-state rate constants of BT2127 (with a k_(cat)/K_m value for pyrophosphate of -1 X 10~5 M~(-1) s~(-1)), together with the gene context, support the assigned in vivo function as an inorganic pyrophosphatase. The X-ray structural analysis of wild-type BT2127 and several variants generated by site-directed mutagenesis shows that substrate discrimination is based, in part, on active sitepan space restrictions imposed by the cap domain (specifically by residues Tyr76 and G1u47). site-directed mutagenesis coupled with 'kinetic analysis of the mutant enzymes identified the residues require, for catalysis, substrate binding, and domain-domain association. On the basis of this structure-function analysis, the catalyti. residues Aspll, Asp13, Thr113, and Lys147 as well the metal binding residues Asp171, Asn172, and G1u47 were used as marker to confirm BT2127 orthologues identified via sequence searches. This bioinformatic analysis demonstrated that the biologica range of BT2127 orthologue is restricted to the phylum Bacteroidetes/Chlorobi. The key structural determinants in th divergence of BT2127 and its closest homologue, β-phosphoglucomutase, control the leaving group size (phosphate vs glucos phosphate) and the position of the Asp acid/base in the open versus closed conformations. HADSF pyrophosphatases represen.
机译：蛋白质序列信息的爆炸要求功能分配的当前策略演变成与基于计算上的函数预测的补充实验方法。这需要基于鉴定特异性决定因素形式的序列标记的策略和更明智的原例定义。在此，我们已经使用集成的生物信息 - 结构机构方法从拟菌ThetaiOraOmicron进行了未知卤代烷烃脱卤素酶超家族成员BT2127（Uniprot登录代码Q8A5V9）的功能分配。 BT2127的底物特异性曲线和Bt2127的稳态速率常数（具有-1×10〜5m〜（-1）〜（-1）的焦磷酸磷酸磷酸的K_（猫）/ K_m值，以及基因背景，支持将分配的体内用作无机焦磷酸酶。野生型BT2127的X射线结构分析和由点定向诱变产生的几种变体表明，部分鉴别部分地基于帽结构域施加的主动参考空间限制（具体通过残留TYR76和G1U47）。与突变酶的动力学分析相结合的位点定向诱变鉴定了残留物需要催化作用，底物结合和结构域域。在该结构函数分析的基础上，催化剂。残留物ASPLL，ASP13，THR113和LYS147以及金属结合残余物ASP171，ASN172和G1U47用作标记物，以确认通过序列搜索鉴定的BT2127邻接。这种生物信息分析表明，BT2127矫正器的Biologica范围仅限于Phylum Bacteropetes / Chorobi。 BT2127的关键结构决定簇及其最近的同源物，β-磷酰型酶，控制离去群体尺寸（磷酸盐与葡糖磷酸盐）和开放与闭合构象的ASP酸/碱的位置。 HADSF焦磷酸酶代表。

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  • 来源
    《Biochemistry》 |2011年第41期|共13页
  • 作者

    Hua Huang; Yury Patskovsky; Rafael Toro; Jeremiah D. Farelli; Chetanya Pandya; Steven C. Almo; Karen N. Allen; Debra Dunaway-Mariano;

  • 作者单位

    Department of Chemistry and Chemical Biology University of New Mexico Albuquerque New Mexico 87131 United States;

    Department of Biochemistry Albert Einstein College of Medicine Jack and Pearl Resnick Campus 1300 Morris Park Avenue Bronx New York 10461 United States;

    Department of Biochemistry Albert Einstein College of Medicine Jack and Pearl Resnick Campus 1300 Morris Park Avenue Bronx New York 10461 United States;

    Department of Chemistry Boston University 590 Commonwealth Avenue Boston Massachusetts 02215-2521 United States;

    Bioinformatics Graduate Program Boston University 24 Cummington Street Boston Massachusetts 02215-2521 United;

    Department of Biochemistry Albert Einstein College of Medicine Jack and Pearl Resnick Campus 1300 Morris Park Avenue Bronx New York 10461 United States;

    Department of Chemistry Boston University 590 Commonwealth Avenue Boston Massachusetts 02215-2521 United States;

    Department of Chemistry and Chemical Biology University of New Mexico Albuquerque New Mexico 87131 United States;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;
  • 关键词

    Structure; Function; thetaiotaomicron;

    机译：结构;功能;Thetaiotaomicron;

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