Crystallographic fragment-binding studies of the Mycobacterium tuberculosis trifunctional enzyme suggest binding pockets for the tails of the acyl-CoA substrates at its active sites and a potential substrate-channeling path between them.

IF 2.6 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS
Subhadra Dalwani, Alexander Metz, Franziska U Huschmann, Manfred S Weiss, Rik K Wierenga, Rajaram Venkatesan
{"title":"Crystallographic fragment-binding studies of the Mycobacterium tuberculosis trifunctional enzyme suggest binding pockets for the tails of the acyl-CoA substrates at its active sites and a potential substrate-channeling path between them.","authors":"Subhadra Dalwani, Alexander Metz, Franziska U Huschmann, Manfred S Weiss, Rik K Wierenga, Rajaram Venkatesan","doi":"10.1107/S2059798324006557","DOIUrl":null,"url":null,"abstract":"<p><p>The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α<sub>2</sub>β<sub>2</sub> tetrameric enzyme in which the α-chain harbors the 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) active sites, and the β-chain provides the 3-ketoacyl-CoA thiolase (KAT) active site. Linear, medium-chain and long-chain 2E-enoyl-CoA molecules are the preferred substrates of MtTFE. Previous crystallographic binding and modeling studies identified binding sites for the acyl-CoA substrates at the three active sites, as well as the NAD binding pocket at the HAD active site. These studies also identified three additional CoA binding sites on the surface of MtTFE that are different from the active sites. It has been proposed that one of these additional sites could be of functional relevance for the substrate channeling (by surface crawling) of reaction intermediates between the three active sites. Here, 226 fragments were screened in a crystallographic fragment-binding study of MtTFE crystals, resulting in the structures of 16 MtTFE-fragment complexes. Analysis of the 121 fragment-binding events shows that the ECH active site is the `binding hotspot' for the tested fragments, with 41 binding events. The mode of binding of the fragments bound at the active sites provides additional insight into how the long-chain acyl moiety of the substrates can be accommodated at their proposed binding pockets. In addition, the 20 fragment-binding events between the active sites identify potential transient binding sites of reaction intermediates relevant to the possible channeling of substrates between these active sites. These results provide a basis for further studies to understand the functional relevance of the latter binding sites and to identify substrates for which channeling is crucial.</p>","PeriodicalId":7116,"journal":{"name":"Acta Crystallographica. Section D, Structural Biology","volume":" ","pages":"605-619"},"PeriodicalIF":2.6000,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11301753/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Acta Crystallographica. Section D, Structural Biology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1107/S2059798324006557","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/7/16 0:00:00","PubModel":"Epub","JCR":"Q2","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0

Abstract

The Mycobacterium tuberculosis trifunctional enzyme (MtTFE) is an α2β2 tetrameric enzyme in which the α-chain harbors the 2E-enoyl-CoA hydratase (ECH) and 3S-hydroxyacyl-CoA dehydrogenase (HAD) active sites, and the β-chain provides the 3-ketoacyl-CoA thiolase (KAT) active site. Linear, medium-chain and long-chain 2E-enoyl-CoA molecules are the preferred substrates of MtTFE. Previous crystallographic binding and modeling studies identified binding sites for the acyl-CoA substrates at the three active sites, as well as the NAD binding pocket at the HAD active site. These studies also identified three additional CoA binding sites on the surface of MtTFE that are different from the active sites. It has been proposed that one of these additional sites could be of functional relevance for the substrate channeling (by surface crawling) of reaction intermediates between the three active sites. Here, 226 fragments were screened in a crystallographic fragment-binding study of MtTFE crystals, resulting in the structures of 16 MtTFE-fragment complexes. Analysis of the 121 fragment-binding events shows that the ECH active site is the `binding hotspot' for the tested fragments, with 41 binding events. The mode of binding of the fragments bound at the active sites provides additional insight into how the long-chain acyl moiety of the substrates can be accommodated at their proposed binding pockets. In addition, the 20 fragment-binding events between the active sites identify potential transient binding sites of reaction intermediates relevant to the possible channeling of substrates between these active sites. These results provide a basis for further studies to understand the functional relevance of the latter binding sites and to identify substrates for which channeling is crucial.

对结核分枝杆菌三官能团酶的晶体片段结合研究表明,在其活性位点上存在酰基-CoA 底物尾部的结合口袋,它们之间可能存在底物通道。
结核分枝杆菌三功能酶(MTTFE)是一种α2β2 四聚体酶,其中α-链含有 2E-enoyl-CoA 水合酶(ECH)和 3S-hydroxyacyl-CoA 脱氢酶(HAD)活性位点,β-链提供 3-Ketoacyl-CoA 硫醇酶(KAT)活性位点。线性、中链和长链 2E-enoyl-CoA 分子是 MtTFE 的首选底物。以前的晶体学结合和建模研究确定了三个活性位点的酰基-CoA 底物结合位点,以及 HAD 活性位点的 NAD 结合袋。这些研究还在 MtTFE 表面发现了与活性位点不同的另外三个 CoA 结合位点。有人提出,其中一个额外位点可能与三个活性位点之间反应中间产物的底物通道(通过表面爬行)功能有关。在此,通过对 MtTFE 晶体进行片段结合研究,筛选出了 226 个片段,从而得到了 16 个 MtTFE 片段复合物的结构。对 121 个片段结合事件的分析表明,ECH 活性位点是测试片段的 "结合热点",有 41 个结合事件。结合在活性位点上的片段的结合模式提供了更多关于底物的长链酰基如何能够容纳在其拟议的结合口袋中的见解。此外,活性位点之间的 20 个片段结合事件确定了与这些活性位点之间可能的底物通道有关的反应中间体的潜在瞬时结合位点。这些结果为进一步研究提供了基础,以便了解后几个结合位点的功能相关性,并确定通道对其至关重要的底物。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
Acta Crystallographica. Section D, Structural Biology
Acta Crystallographica. Section D, Structural Biology BIOCHEMICAL RESEARCH METHODSBIOCHEMISTRY &-BIOCHEMISTRY & MOLECULAR BIOLOGY
CiteScore
4.50
自引率
13.60%
发文量
216
期刊介绍: Acta Crystallographica Section D welcomes the submission of articles covering any aspect of structural biology, with a particular emphasis on the structures of biological macromolecules or the methods used to determine them. Reports on new structures of biological importance may address the smallest macromolecules to the largest complex molecular machines. These structures may have been determined using any structural biology technique including crystallography, NMR, cryoEM and/or other techniques. The key criterion is that such articles must present significant new insights into biological, chemical or medical sciences. The inclusion of complementary data that support the conclusions drawn from the structural studies (such as binding studies, mass spectrometry, enzyme assays, or analysis of mutants or other modified forms of biological macromolecule) is encouraged. Methods articles may include new approaches to any aspect of biological structure determination or structure analysis but will only be accepted where they focus on new methods that are demonstrated to be of general applicability and importance to structural biology. Articles describing particularly difficult problems in structural biology are also welcomed, if the analysis would provide useful insights to others facing similar problems.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信