Lignin Degradation by Phanerochaete Chrysosporium: Biochemical Characterization of Extracellular Peroxidases

本项目的总体目的是进一步了解降解木质素的黑孢Phanerochaete chrysosporium的锰过氧化物酶（MnP）和木质素过氧化物酶（LiP）。具体目标是表征这些酶的结构、机制和功能。利用PI开发的同源表达系统，他们计划构建、表达和表征几个位点导向的MnP突变体。这些突变包括Arg177（一种与Mn配体Glu35相互作用的氨基酸残基）的几个突变，以及Mn结合位点残基的其他突变。它们将决定这些突变对MnP功能的影响。他们还计划继续用生物物理方法表征MnP Phe190突变体，并构建和表征MnP Phe45突变体。为了更好地理解LiP是如何氧化聚合木质素的，我们将研究木质素模型聚合物底物核糖核酸酶的LiP氧化。特别地，将研究戊曲醇在该反应中的作用。利用一系列二甲氧基苄基醇LiP底物，PI将确定在LiP反应中形成和稳定阳离子自由基中间体的要求。木质纤维素占陆地生物量的95%，木质素的存在大大延缓了微生物对纤维素的分解；因此，木质素在地球的碳循环中起着关键作用，是各种生物质利用生物技术的核心。本文对自然界中主要催化木质素生物降解的真菌酶的结构和机制进行了研究。这项工作具有重要的生态和生物技术应用价值。

9808430 Gold The overall aim of this project is to further understanding of manganese peroxidase (MnP) and lignin peroxidase (LiP) from the lignin-degrading basidiomycete Phanerochaete chrysosporium. The specific objectives are to characterize the structures, mechanisms, and functions of these enzymes. Using a homologous expression system developed by the PI, they plan to construct, express, and characterize several site-directed mutants of MnP. These include several mutations at Arg177, an amino acid residue which interacts with the Mn ligand Glu35, and additional mutations of Mn bindidng site residues. They will determine the effects of these mutations on the function of MnP. They also plan to continue to characterize MnP Phe190 mutants by biophysical methods and to construct and characterize MnP Phe45 mutants. To better understand how LiP oxidizes polymeric lignin, the LiP oxidation of a lignin model polymeric substrate, ribonuclease, will be examined. In particular, the role of veratryl alcohol in this reaction will be examined. Using a series of dimethoxybenzyl alcohol LiP substrates, the PI will determine the requirements for forming and stablizing cation radical intermediates in the LiP reaction. Lignocellulose comprises-95% of terrestrial biomass, and the presence of lignin greatly retards the microbial breakdown of cellulose; thus, lignin plays a key role in the earth's carbon cycle and is central to a wide variety of biotechnologies for biomass utilization. This proposal focuses on several unresolved questions concerning the structures and mechanisms of the fungal enzymes which are chiefly responsible for catalyzing lignin biodegradation in nature. The proposed work has important ecological and biotechnological applications.