{"title":"Breaking the Myth of Enzymatic Azoreduction.","authors":"Yu-Ju Peng, Bing Xu, Steven E Rokita","doi":"10.1021/acschembio.4c00779","DOIUrl":null,"url":null,"abstract":"<p><p>Flavin-dependent azoreductases have been applied to a wide range of tasks from decolorizing numerous azo dyes to releasing azo-conjugated prodrugs. A general narrative reiterated in much of the literature suggests that this enzyme promotes sequential reduction of both the azo-containing substrate and its corresponding hydrazo product to release the aryl amine components while consuming two equivalents of NAD(P)H. Indeed, such aryl amines can be formed by incubation of certain azo compounds with azoreductases, but the nature of the substrates capable of this apparent azo bond lysis remained unknown. We have now prepared a set of azobenzene derivatives and characterized their turnover and products after treatment with azoreductase from <i>Escherichia coli</i> to discover the structural basis regulating aryl amine formation. Without resonance donation by aryl substituents, reduction ceases at the hydrazo product. This indicates that azoreductases do not act on the hydrazo bond. Instead, aryl amine formation depends on a spontaneous hydrazo bond lysis that is promoted by resonance stabilization and subsequent reduction of the quinone-like intermediate by azoreductase. Experimental and computational approaches confirm the substituent dependence of this process. With knowledge of this requirement, full release of aryl amines from azo-conjugates can now be designed and applied with confidence.</p>","PeriodicalId":11,"journal":{"name":"ACS Chemical Biology","volume":" ","pages":""},"PeriodicalIF":3.5000,"publicationDate":"2024-12-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Chemical Biology","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1021/acschembio.4c00779","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Flavin-dependent azoreductases have been applied to a wide range of tasks from decolorizing numerous azo dyes to releasing azo-conjugated prodrugs. A general narrative reiterated in much of the literature suggests that this enzyme promotes sequential reduction of both the azo-containing substrate and its corresponding hydrazo product to release the aryl amine components while consuming two equivalents of NAD(P)H. Indeed, such aryl amines can be formed by incubation of certain azo compounds with azoreductases, but the nature of the substrates capable of this apparent azo bond lysis remained unknown. We have now prepared a set of azobenzene derivatives and characterized their turnover and products after treatment with azoreductase from Escherichia coli to discover the structural basis regulating aryl amine formation. Without resonance donation by aryl substituents, reduction ceases at the hydrazo product. This indicates that azoreductases do not act on the hydrazo bond. Instead, aryl amine formation depends on a spontaneous hydrazo bond lysis that is promoted by resonance stabilization and subsequent reduction of the quinone-like intermediate by azoreductase. Experimental and computational approaches confirm the substituent dependence of this process. With knowledge of this requirement, full release of aryl amines from azo-conjugates can now be designed and applied with confidence.
期刊介绍:
ACS Chemical Biology provides an international forum for the rapid communication of research that broadly embraces the interface between chemistry and biology.
The journal also serves as a forum to facilitate the communication between biologists and chemists that will translate into new research opportunities and discoveries. Results will be published in which molecular reasoning has been used to probe questions through in vitro investigations, cell biological methods, or organismic studies.
We welcome mechanistic studies on proteins, nucleic acids, sugars, lipids, and nonbiological polymers. The journal serves a large scientific community, exploring cellular function from both chemical and biological perspectives. It is understood that submitted work is based upon original results and has not been published previously.