Extracellular alpha/beta-hydrolase from Paenibacillus species shares structural and functional homology to tobacco salicylic acid binding protein 2.

An alpha/ beta hydrolase annotated as a putative salicylate esterase within the genome of a species of Paenibacillus previously identified from differential and selective growth on Kraft lignin was structurally and functionally characterised. Feruloyl esterases are key to the degradation of lignin in several bacterial species and although this activity was investigated, no such activity was observed. The crystal structure of the Paenibacillus esterase, here denoted as PnbE, was determined at 1.32 Å resolution, showing high similarity to Nicotiana tabacum salicylic acid binding protein 2 from the protein database. Structural similarities between these two structures across the core domains and key catalytic residues were observed, with superposition of catalytic residues giving an RMSD of 0.5 Å across equivalent Cα atoms. Conversely, the cap domains of PnbE and Nicotiana tabacum SABP2 showed greater divergence with decreased flexibility in the PnbE cap structure. Activity of PnbE as a putative methyl salicylate esterase was supported with binding studies showing affinity for salicylic acid and functional studies showing methyl salicylate esterase activity. We hypothesise that this activity could enrich Paenibacillus sp. within the rhizosphere by increasing salicylic acid concentrations within the soil.

Protein engineering of Pseudomonas fluorescens peroxidase Dyp1B for oxidation of phenolic and polymeric lignin substrates.

Directed evolution was applied to dye-decolourizing peroxidase Dyp1B from Pseudomonas fluorescens Pf-5, in order to enhance the activity for oxidation of phenolic and lignin substrates. Saturation mutagenesis was used to generate focused libraries at 7 active site residues in the vicinity of the heme cofactor, and the libraries were screened for activity towards 2,6-dichlorophenol. Mutants N193 L and H169 L were found to show 7-8 fold enhanced kcat/KM towards DCP, and replacements at Val205 and Ala209 also showed enhanced activity towards alkali Kraft lignin. Residues near the predicted Mn(II) binding site were also investigated by site-directed mutagenesis, and mutants S223 N and H127R showed 4-7-fold increased kcat/KM for Mn(II) oxidation. Mutant F128R also showed enhanced thermostability, compared to wild-type Dyp1B. Testing of mutants for low molecular weight product release from Protobind alkali lignin revealed that mutant H169 L showed enhanced product release, compared with WT enzyme, and the formation of three low molecular weight metabolites by this mutant was detected by reverse phase HPLC analysis.