Stimulation of Lignocellulosic Biomass Hydrolysis by Proteins of Glycoside Hydrolase Family 61: Structure and Function of a Large, Enigmatic Family

摘要

Currently, the relatively high cost of enzymes such as glycoside hydrolases that catalyze cellulosenhydrolysis represents a barrier to commercialization of a biorefinery capable of producing renewablentransportable fuels such as ethanol from abundant lignocellulosic biomass. Among the many families ofnglycoside hydrolases that catalyze cellulose and hemicellulose hydrolysis, few are more enigmatic than familyn61 (GH61), originally classified based on measurement of very weak endo-1,4-β-D-glucanase activity in onenfamily member. Here we show that certain GH61 proteins lack measurable hydrolytic activity by themselvesnbut in the presence of various divalent metal ions can significantly reduce the total protein loading required tonhydrolyze lignocellulosic biomass. We also solved the structure of one highly active GH61 protein and findnthat it is devoid of conserved, closely juxtaposed acidic side chains that could serve as general proton donornand nucleophile/base in a canonical hydrolytic reaction, and we conclude that theGH61 proteins are unlikelynto be glycoside hydrolases. Structure-based mutagenesis shows the importance of several conserved residuesnfor GH61 function. By incorporating the gene for one GH61 protein into a commercial Trichoderma reeseinstrain producing high levels of cellulolytic enzymes, we are able to reduce by 2-fold the total protein loadingn(and hence the cost) required to hydrolyze lignocellulosic biomass.