The structure and mode of action of Caldicellulosiruptor bescii family 3 pectate lyase in biomass deconstruction.

The unique active site of the Caldicellulosiruptor bescii family 3 pectate lyase catalytic module (PL3-cat) has been structurally described and synergistic digestion studies with C. bescii cellulase A have been performed on unpretreated biomass. The X-ray structure of PL3-cat was determined at 1.6 Å resolution (PDB entry 4ew9) in complex with the products of trigalacturonic acid. Comparison with family 1 pectate lyase (PL1) structures shows that the active site of the PL3 catalytic module is considerably different. However, on superimposing the identical sugar rings at the -2 subsites conserved interactions could be identified. Interestingly, only one catalytic residue, the lysine that donates the proton to the carboxylate group in the ß-elimination reaction of PL1 (Lys108 in PL3-cat), is 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 with other proton-abstraction reactions reveals that in PL3 the α-proton is abstracted by a lysine, in a striking similarity to enolases. These observations led 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 ß-elimination reaction by protonating the O4 atom of the substrate. Also, our digestion experiments with unpretreated switchgrass show that the loadings of C. bescii cellobiohydrolase A (CelA) can be lowered by the 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.

Identification and characterization of novel catalytic bioscavengers of organophosphorus nerve agents.

In an effort to discover novel catalytic bioscavengers of organophosphorus (OP) nerve agents, cell lysates from a diverse set of bacterial strains were screened for their capacity to hydrolyze the OP nerve agents VX, VR, and soman (GD). The library of bacterial strains was identified using both random and rational approaches. Specifically, two representative strains from eight categories of extremophiles were chosen at random. For the rational approach, the protein sequence of organophosphorus hydrolase (OPH) from Brevundimonas diminuta was searched against a non-redundant protein database using the Basic Local Alignment Search Tool to find regions of local similarity between sequences. Over 15 protein sequences with significant sequence similarity to OPH were identified from a variety of bacterial strains. Some of these matches were based on predicted protein structures derived from bacterial genome sequences rather than from bona fide proteins isolated from bacteria. Of the 25 strains selected for nerve agent testing, three bacterial strains had measurable levels of OP hydrolase activity. These strains are Ammoniphilus oxalaticus, Haloarcula sp., and Micromonospora aurantiaca. Lysates from A. oxalaticus had detectable hydrolysis of VR; Haloarcula sp. had appreciable hydrolysis of VX and VR, whereas lysates from M. aurantiaca had detectable hydrolysis of VR and GD.