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

Abstract

Currently, the relatively high cost of enzymes such as glycoside hydrolases that catalyze cellulose hydrolysis represents a barrier to commercialization of a biorefinery capable of producing renewable transportable fuels such as ethanol from abundant lignocellulosic biomass. Among the many families of glycoside hydrolases that catalyze cellulose and hemicellulose hydrolysis, few are more enigmatic than family 61 (GH61), originally classified based on measurement of very weak endo-1,4-beta-d-glucanase activity in one family member. Here we show that certain GH61 proteins lack measurable hydrolytic activity by themselves but in the presence of various divalent metal ions can significantly reduce the total protein loading required to hydrolyze lignocellulosic biomass. We also solved the structure of one highly active GH61 protein and find that it is devoid of conserved, closely juxtaposed acidic side chains that could serve as general proton donor and nucleophile/base in a canonical hydrolytic reaction, and we conclude that the GH61 proteins are unlikely to be glycoside hydrolases. Structure-based mutagenesis shows the importance of several conserved residues for GH61 function. By incorporating the gene for one GH61 protein into a commercial Trichoderma reesei strain producing high levels of cellulolytic enzymes, we are able to reduce by 2-fold the total protein loading (and hence the cost) required to hydrolyze lignocellulosic biomass.

Cleavage of cellulose by a CBM33 protein

“…However, the potentiating mechanism of these proteins remains enigmatic and activity has so far only been shown for rather complex substrates, that is, not for pure cellulose. 17 In this study, we demonstrate that CelS2 from Streptomyces coelicolor A3(2), a two-domain protein consisting of a 194-residue CBM33 supplemented with a well-known 99-residue cellulose-binding domain (CBM2), indeed is capable of cellulose cleavage, generating oxidized chain ends and boosting cellulase activity. This is the first time such an activity is experimentally shown.…”

“…However, several metals worked well, confirming the apparent promiscuity that has been observed in other studies of both CBM33s 11 and GH61s. 17 Purified CelS2 retained considerable activity without the addition of metals and it is likely that this activity is due to high affinity binding of another as yet unidentified metal. Addition of ethylenediaminetetraacetic acid (EDTA) inhibited the enzyme.…”

“…12 These results reveal a new paradigm for enzymatic cellulose conversion and perhaps for enzymatic conversion of polysaccharides in general. Proteins classified as GH61 seem to be the fungal counterpart of the bacterial CBM33 proteins, as they are structurally similar and have a potentiating effect on cellulases, 17 although their mechanism remains unknown. The fact that such a ''cellulaseboosting activity'' not only occurs in fungi (GH61) but also in bacteria (CBM33) indicates the importance of this activity in nature.…”

“…Another important issue for future studies is the role of the divalent metal ion which remains somewhat enigmatic for both CBM33 and GH61 enzymes. 11,17,18 We have used Mg 2þ in this study because an initial screening showed high activity when this metal was added. However, several metals worked well, confirming the apparent promiscuity that has been observed in other studies of both CBM33s 11 and GH61s.…”

“…Indeed, several bacteria that are able to degrade crystalline cellulose contain multiple CBM33 proteins 13,14 and some of these are known to be coregulated with cellulases. 15,16 Proteins classified as glycoside hydrolase family 61 (GH61) are structurally similar to CBM33 17,18 and are known to act synergistically with cellulases. However, the potentiating mechanism of these proteins remains enigmatic and activity has so far only been shown for rather complex substrates, that is, not for pure cellulose.…”