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.

The structure of the DNA-binding NAC domain of Arabidopsis ANAC (abscisic-acid-responsive NAC) has been determined by Xray crystallography to 1.9 Å resolution (Protein Data Bank codes 1UT4 and 1UT7). This is the first structure determined for a member of the NAC family of plant-specific transcriptional regulators. NAC proteins are characterized by their conserved N-terminal NAC domains that can bind both DNA and other proteins. NAC proteins are involved in developmental processes, including formation of the shoot apical meristem, floral organs and lateral shoots, as well as in plant hormonal control and defence. The NAC domain does not possess a classical helix-turnhelix motif; instead it reveals a new transcription factor fold consisting of a twisted b-sheet surrounded by a few helical elements. The functional dimer formed by the NAC domain was identified in the structure, which will serve as a structural template for understanding NAC protein function at the molecular level.

Structural and sequence comparisons have revealed that, despite very low sequence similarities, RGAE is related to seven other proteins. They are all members of a new hydrolase family, the SGNH-hydrolase family, which includes the carbohydrate esterase family 12 as a distinct subfamily. The SGNH-hydrolase family is characterised by having four conserved blocks of residues, each with one completely conserved residue; serine, glycine, asparagine and histidine, respectively. Each of the four residues plays a role in the catalytic function.

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