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Discovery of new enzymes and metabolic pathways by using structure and genome context

机译：通过使用结构和基因组背景发现新的酶和代谢途径

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摘要

Assigning valid functions to proteins identified in genome projects is challenging: overprediction and database annotation errors are the principal concerns. We and others are developing computationguided strategies for functional discovery with 'metabolite docking' to experimentally derived or homology-based three-dimensional structures. Bacterial metabolic pathways often are encoded by 'genome neighbourhoods' (gene clusters and/or operons), which can provide important clues for functional assignment. We recentiy demonstrated the synergy of docking and pathway context by 'predicting' the intermediates in the glycolytic pathway in Escherichia coli. Metabolite docking to multiple binding proteins and enzymes in the same pathway increases the reliability of in silico predictions of substrate specificities because the pathway intermediates are structurally similar. Here we report that structure-guided approaches for predicting the substrate specificities of several enzymes encoded by a bacterial gene cluster allowed the correct prediction of the in vitro activity of a structurally characterized enzyme of unknown function (PDB 2PMQ), 2-epimerization of trans-4-hydroxy-L-proline betaine (tHyp-B) and cis-4-hydroxy-D-proline betaine (cHyp-B), and also the correct identification of the catabolic pathway in which Hyp-B 2-epimerase participates. The substrate-liganded pose predicted by virtual library screening (docking) was confirmed experimentally. The enzymatic activities in the predicted pathway were confirmed by in vitro assays and genetic analyses; the intermediates were identified by metabolomics; and repression of the genes encoding the pathway by high salt concentrations was established by transcriptomics, confirming the osmolyte role of tHyp-B. This study establishes the utility of structure-guided functional predictions to enable the discovery of new metabolic pathways.
机译：为基因组项目中鉴定出的蛋白质分配有效功能是一项挑战：过度预测和数据库注释错误是主要问题。我们和其他人正在开发功能导向的计算指导策略，其中包括“代谢物对接”到实验衍生或基于同源性的三维结构。细菌代谢途径通常由“基因组社区”（基因簇和/或操纵子）编码，这可以为功能分配提供重要线索。我们最近通过“预测”大肠杆菌糖酵解途径中的中间体证明了对接和途径背景的协同作用。代谢物与同一途径中的多种结合蛋白和酶对接，因为途径中间体在结构上相似，从而提高了计算机模拟底物特异性的可靠性。在这里，我们报告了一种结构指导的方法，用于预测由细菌基因簇编码的几种酶的底物特异性，可以正确预测功能未知的结构特征化酶（PDB 2PMQ）的体外活性，反式2- 4-羟基-L-脯氨酸甜菜碱（tHyp-B）和顺式-4-羟基-D-脯氨酸甜菜碱（cHyp-B），以及对Hyp-B2-表异构酶参与的分解代谢途径的正确鉴定。实验证实了通过虚拟文库筛选（对接）预测的底物配体姿势。通过体外测定和遗传分析证实了预测途径中的酶活性。通过代谢组学鉴定中间体；转录组学证实了高盐浓度下编码该途径的基因的抑制，证实了tHyp-B的渗透液作用。这项研究建立了结构指导的功能预测的实用程序，以使能够发现新的代谢途径。

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  • 来源
    《Nature》 |2013年第7473期|698-702|共5页
  • 作者

    Suwen Zhao; Ritesh Kumar; Ayano Sakai; Matthew W. Vetting; B. McKay Wood; Shoshana Brown; Jeffery B. Bonanno; Brandan S. Hillerich; Ronald D. Seidel; Patricia C. Babbitt; Steven C. Almo; Jonathan V. Sweedler; John A. Gerlt; John E. Cronan; Matthew P. Jacobson;

  • 作者单位

    Departmentof Pharmaceutical Chemistry, University of California, San Francisco, California 94143, USA;

    Institute for Genomic Biology, University of Illinois at Urbana-Champaign.Urbana, Illinois 61801,USA;

    Institute for Genomic Biology, University of Illinois at Urbana-Champaign.Urbana, Illinois 61801,USA;

    Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA;

    Institute for Genomic Biology, University of Illinois at Urbana-Champaign.Urbana, Illinois 61801,USA;

    Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco,California 94143, USA;

    Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA;

    Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA;

    Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA;

    Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco,California 94143, USA;

    Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA;

    Institute for Genomic Biology, University of Illinois at Urbana-Champaign.Urbana, Illinois 61801,USA,Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA;

    Institute for Genomic Biology, University of Illinois at Urbana-Champaign.Urbana, Illinois 61801,USA,Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA,Department of Biochemistry, University of Illinois at Urbana-Champaign,Urbana, Illinois 61801, USA;

    Institute for Genomic Biology, University of Illinois at Urbana-Champaign.Urbana, Illinois 61801,USA,Department of Biochemistry, University of Illinois at Urbana-Champaign,Urbana, Illinois 61801, USA,Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA;

    Departmentof Pharmaceutical Chemistry, University of California, San Francisco, California 94143, USA;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
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