Monday, April 29, 2013
Exhibit Hall
We have described structurally the unique active site of Caldicellulosiruptor bescii family 3 pectate lyase catalytic module (PL3-cat) and performed synergistic digestion studies together with C. bescii cellulase A on unpretreated biomass. The X-ray structure of PL3-cat was determined in complex with the products of trigalacturonic acid. Comparison to family 1 pectate lyase structures shows that the active site of PL3 catalytic module is considerably different. By superimposing the identical sugar rings at the -2 subsites we were able to identify conserved interactions. Interestingly, only one catalytic residue, the lysine that donates the proton to the carboxylate group in the β-elimination reaction of PL1, has been conserved in PL3 and there is no arginine to abstract the proton from the c5 carbon of the galactouronate ring. This suggests that the reaction mechanism of PL3 requires different catalytic residues. Most interestingly, comparison to other proton abstractions reactions reveals that in PL3 the α-proton is abstracted by a lysine in a striking similarity to enolases. These observations lead us to propose that in PL3-cat, Lys108 is the catalytic base, Glu84 is the catalytic acid, and an acidified water molecule completes the anti β-elimination reaction by protonating the O4 oxygen-atom of the substrate. Also, our digestion experiments with unpretreated switchgrass show that the loadings of C. bescii CelA can be lowered by addition of PL3 to the reaction mixture. This result suggests that PL3 can significantly improve the deconstruction of unpretreated biomass by allowing other enzymes to better access their preferred substrates.