Lignin Degradation by Phanerochaete Chrysosporium: Biochemical Characterization of Extracellular Peroxidases

9808430 Gold本项目的总体目标是进一步了解来自木质素降解担子菌黄孢原毛革菌的锰过氧化物酶（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。 使用PI开发的同源表达系统，他们计划构建，表达和表征MNP的几种定点突变体。 这些包括Arg 177处的几个突变，Arg 177是与Mn配体Glu 35相互作用的氨基酸残基，以及Mn结合位点残基的额外突变。 他们将确定这些突变对MNP功能的影响。 他们还计划继续通过生物物理方法表征MnP Phe 190突变体，并构建和表征MnP Phe 45突变体。 为了更好地理解LiP如何氧化聚合木质素，将检查木质素模型聚合物底物核糖核酸酶的LiP氧化。 特别是，藜芦醇在该反应中的作用将被检查。使用一系列二甲氧基苄醇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。 木质素纤维素占陆地生物质的约95%，并且木质素的存在极大地阻碍了纤维素的微生物分解;因此，木质素在地球的碳循环中起关键作用，并且是用于生物质利用的各种生物技术的核心。 本文主要讨论了在自然界中催化木质素生物降解的真菌酶的结构和作用机制。 拟议的工作具有重要的生态和生物技术应用。

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.