Lignin Degradation by Phanerochaete Chrysosporium: Biochemical Characterization of Extracellular Peroxidases

9808430金这个项目的总体目标是进一步了解降解木质素的担子菌黄孢原毛平革菌的锰过氧化物酶和木质素过氧化物酶。具体目标是描述这些酶的结构、机制和功能。利用PI开发的同源表达系统，他们计划构建、表达和鉴定MNP的几个定点突变体。这些突变包括Arg177处的几个突变，这是一种与锰配体Glu35相互作用的氨基酸残基，以及锰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。他们还计划继续用生物物理方法鉴定MNP Phe190突变体，并构建和鉴定MNP Phe45突变体。为了更好地了解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。特别是，藜芦醇在该反应中的作用将被研究。使用一系列二甲氧基苯甲醇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%，而木质素的存在极大地延缓了纤维素的微生物分解；因此，木质素在地球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。这项建议集中在几个悬而未决的问题，关于真菌酶的结构和机制，主要负责催化木质素在自然界中的生物降解。拟议的工作具有重要的生态学和生物技术应用。

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.