Structural and Biochemical Insights into Lignin-Oxidizing Activity of Bacterial Peroxidases against Soluble Substrates and Kraft Lignin.

IF 3.5 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Zahra Choolaei, Anna N Khusnutdinova, Tatiana Skarina, Peter Stogios, Patrick Diep, Sofia Lemak, Elizabeth A Edwards, Alexei Savchenko, Alexander F Yakunin
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引用次数: 0

Abstract

Great interest has recently been drawn to the production of value-added products from lignin; however, its recalcitrance and high chemical complexity have made this challenging. Dye-decolorizing peroxidases and catalase-peroxidases are among the enzymes that are recognized to play important roles in environmental lignin oxidation. However, bacterial lignin-oxidizing enzymes remain less characterized compared to related proteins from fungi. In this study, screening of 18 purified bacterial peroxidases against the general chromogenic substrate 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) revealed the presence of peroxidase activity in all proteins. Agarose plate-based screens with kraft lignin identified detectable and high lignin oxidation activity in 15 purified proteins. Crystal structures were determined for the DyP-type peroxidases FC2591 from Frankia casuarinae, PF3257 from Pseudomonas fluorescens, and PR9465 from Pseudomonas rhizosphaerae. The structures revealed the presence of hemes with bound oxygens coordinated by conserved His, Arg, and Asp residues as well as three molecular tunnels connecting the heme with the protein surface. Structure-based site-directed mutagenesis of FC2591 identified at least five active site residues as essential for oxidase activity against both ABTS and lignin, whereas the S370A mutant protein showed a three- to 4-fold activity increase with both substrates. HPLC analysis of reaction products of the wild-type FC2591 and S370A mutant proteins with the model lignin dimer guaiacylglycerol-β-guaiacyl ether and kraft lignin revealed the formation of products consistent with the radical coupling of the reaction intermediates. Thus, this study identified novel bacterial heme peroxidases with lignin oxidation activity and provided further insights into our understanding of these enzymes.

细菌过氧化物酶对可溶性底物和硫酸盐木质素氧化活性的结构和生化研究。
最近,人们对从木质素中生产增值产品产生了极大的兴趣;然而,它的顽固性和高化学复杂性使其具有挑战性。染料脱色过氧化物酶和过氧化氢酶过氧化物酶是公认的在环境木质素氧化中起重要作用的酶。然而,与真菌中的相关蛋白相比,细菌木质素氧化酶的特征仍然较少。在这项研究中,筛选了18种纯化的细菌过氧化物酶对抗一般显色底物2,2'-azinobis(3-乙基苯并噻唑啉-6-磺酸盐)(ABTS),结果显示所有蛋白质都存在过氧化物酶活性。基于琼脂糖板的硫酸盐木质素筛选鉴定了15种纯化蛋白的可检测和高木质素氧化活性。测定了木麻黄假单胞菌中的dp型过氧化物酶FC2591、荧光假单胞菌中的PF3257和根孢假单胞菌中的PR9465的晶体结构。这些结构揭示了血红素与由保守的His、Arg和Asp残基协调的结合氧以及连接血红素与蛋白质表面的三个分子通道的存在。对FC2591进行的基于结构的位点定向诱变发现,至少有5个活性位点残基是氧化酶对ABTS和木质素的活性所必需的,而S370A突变蛋白对这两种底物的活性都增加了3到4倍。对野生型FC2591和S370A突变蛋白与木质素二聚体愈创木酰甘油-β-愈创木酰醚和硫酸盐木质素的反应产物进行HPLC分析,发现产物的形成符合反应中间体的自由基偶联。因此,本研究发现了具有木质素氧化活性的新型细菌血红素过氧化物酶,并为我们对这些酶的理解提供了进一步的见解。
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来源期刊
ACS Chemical Biology
ACS Chemical Biology 生物-生化与分子生物学
CiteScore
7.50
自引率
5.00%
发文量
353
审稿时长
3.3 months
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.
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