Mst Sharmin Aktar, Vesna de Serrano, Reza A. Ghiladi and Stefan Franzen*,
{"title":"Structural Comparison of Substrate Binding Sites in Dehaloperoxidase A and B","authors":"Mst Sharmin Aktar, Vesna de Serrano, Reza A. Ghiladi and Stefan Franzen*, ","doi":"10.1021/acs.biochem.4c00179","DOIUrl":null,"url":null,"abstract":"<p >Dehalperoxidase (DHP) has diverse catalytic activities depending on the substrate binding conformation, pH, and dynamics in the distal pocket above the heme. According to our hypothesis, the molecular structure of the substrate and binding orientation in DHP guide enzymatic function. Enzyme kinetic studies have shown that the catalytic activity of DHP B is significantly higher than that of DHP A despite 96% sequence homology. There are more than 30 substrate-bound structures with DHP B, each providing insight into the nature of enzymatic binding at the active site. By contrast, the only X-ray crystallographic structures of small molecules in a complex with DHP A are phenols. This study is focused on investigating substrate binding in DHP A to compare with DHP B structures. Fifteen substrates were selected that were known to bind to DHP B in the crystal to test whether soaking substrates into DHP A would yield similar structures. Five of these substrates yielded X-ray crystal structures of substrate-bound DHP A, namely, 2,4-dichlorophenol (1.48 Å, PDB: 8EJN), 2,4-dibromophenol (1.52 Å, PDB: 8VSK), 4-nitrophenol (2.03 Å, PDB: 8VKC), 4-nitrocatechol (1.40 Å, PDB: 8VKD), and 4-bromo-o-cresol (1.64 Å, PDB: 8VZR). For the remaining substrates that bind to DHP B, such as cresols, 5-bromoindole, benzimidazole, 4,4-biphenol, 4.4-ethylidenebisphenol, 2,4-dimethoxyphenol, and guaiacol, the electron density maps in DHP A are not sufficient to determine the presence of the substrates, much less their orientation. In our hands, only phenols, 4-Br-o-cresol, and 4-nitrocatechol can be soaked into crystalline DHP A. None of the larger substrates were observed to bind. A minimum of seven hanging drops were selected for soaking with more than 50 crystals screened for each substrate. The five high-quality examples of direct comparison of modes of binding in DHP A and B for the same substrate provide further support for the hypothesis that the substrate-binding conformation determines the enzyme function of DHP.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":null,"pages":null},"PeriodicalIF":2.9000,"publicationDate":"2024-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochemistry Biochemistry","FirstCategoryId":"1","ListUrlMain":"https://pubs.acs.org/doi/10.1021/acs.biochem.4c00179","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Dehalperoxidase (DHP) has diverse catalytic activities depending on the substrate binding conformation, pH, and dynamics in the distal pocket above the heme. According to our hypothesis, the molecular structure of the substrate and binding orientation in DHP guide enzymatic function. Enzyme kinetic studies have shown that the catalytic activity of DHP B is significantly higher than that of DHP A despite 96% sequence homology. There are more than 30 substrate-bound structures with DHP B, each providing insight into the nature of enzymatic binding at the active site. By contrast, the only X-ray crystallographic structures of small molecules in a complex with DHP A are phenols. This study is focused on investigating substrate binding in DHP A to compare with DHP B structures. Fifteen substrates were selected that were known to bind to DHP B in the crystal to test whether soaking substrates into DHP A would yield similar structures. Five of these substrates yielded X-ray crystal structures of substrate-bound DHP A, namely, 2,4-dichlorophenol (1.48 Å, PDB: 8EJN), 2,4-dibromophenol (1.52 Å, PDB: 8VSK), 4-nitrophenol (2.03 Å, PDB: 8VKC), 4-nitrocatechol (1.40 Å, PDB: 8VKD), and 4-bromo-o-cresol (1.64 Å, PDB: 8VZR). For the remaining substrates that bind to DHP B, such as cresols, 5-bromoindole, benzimidazole, 4,4-biphenol, 4.4-ethylidenebisphenol, 2,4-dimethoxyphenol, and guaiacol, the electron density maps in DHP A are not sufficient to determine the presence of the substrates, much less their orientation. In our hands, only phenols, 4-Br-o-cresol, and 4-nitrocatechol can be soaked into crystalline DHP A. None of the larger substrates were observed to bind. A minimum of seven hanging drops were selected for soaking with more than 50 crystals screened for each substrate. The five high-quality examples of direct comparison of modes of binding in DHP A and B for the same substrate provide further support for the hypothesis that the substrate-binding conformation determines the enzyme function of DHP.
脱氢过氧化物酶(DHP)具有不同的催化活性,这取决于底物的结合构象、pH 值以及血红素上方远端口袋中的动态变化。根据我们的假设,底物的分子结构和在 DHP 中的结合方向会引导酶的功能。酶动力学研究表明,尽管序列同源性高达 96%,但 DHP B 的催化活性明显高于 DHP A。DHP B 有 30 多种底物结合结构,每种结构都能让人深入了解活性位点酶结合的性质。相比之下,与 DHP A 复合物的小分子 X 射线晶体结构中只有苯酚。本研究的重点是研究 DHP A 与底物的结合,并与 DHP B 的结构进行比较。研究人员选择了已知能与晶体中的 DHP B 结合的 15 种底物,以测试将底物浸泡到 DHP A 中是否会产生类似的结构。其中五种底物产生了与底物结合的 DHP A 的 X 射线晶体结构,即 2,4-二氯苯酚(1.48 Å,PDB:8EJN)、2,4-二溴苯酚(1.52 Å,PDB:8VSK)、4-硝基苯酚(2.03 Å,PDB:8VKC)、4-硝基邻苯二酚(1.40 Å,PDB:8VKD)和 4-溴邻甲酚(1.64 Å,PDB:8VZR)。对于与 DHP B 结合的其余底物,如甲酚、5-溴吲哚、苯并咪唑、4,4-联苯酚、4.4-亚乙基双酚、2,4-二甲氧基苯酚和愈创木酚,DHP A 中的电子密度图不足以确定底物的存在,更不用说它们的方向了。在我们的研究中,只有苯酚、4-Br-邻甲酚和 4-硝基邻苯二酚可以浸入结晶的 DHP A 中。每种基质至少选择 7 个悬滴进行浸泡,筛选出 50 个以上的晶体。这五个直接比较 DHP A 和 B 与相同底物结合模式的高质量实例进一步支持了底物结合构象决定 DHP 酶功能的假设。
期刊介绍:
Biochemistry provides an international forum for publishing exceptional, rigorous, high-impact research across all of biological chemistry. This broad scope includes studies on the chemical, physical, mechanistic, and/or structural basis of biological or cell function, and encompasses the fields of chemical biology, synthetic biology, disease biology, cell biology, nucleic acid biology, neuroscience, structural biology, and biophysics. In addition to traditional Research Articles, Biochemistry also publishes Communications, Viewpoints, and Perspectives, as well as From the Bench articles that report new methods of particular interest to the biological chemistry community.