ABSTRACT
BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) opened a new horizon for biomass deconstruction. They use a redox mechanism not yet fully understood and the range of substrates initially envisaged to be the crystalline polysaccharides is steadily expanding to non-crystalline ones. RESULTS: The enzyme KpLPMO10A from the actinomycete Kitasatospora papulosa was cloned and overexpressed in Escherichia coli cells in the functional form with native N-terminal. The enzyme can release oxidized species from chitin (C1-type oxidation) and cellulose (C1/C4-type oxidation) similarly to other AA10 members from clade II (subclade A). Interestingly, KpLPMO10A also cleaves isolated xylan (not complexed with cellulose, C4-type oxidation), a rare activity among LPMOs not described yet for the AA10 family. The synergistic effect of KpLPMO10A with Celluclast® and an endo-ß-1,4-xylanase also supports this finding. The crystallographic elucidation of KpLPMO10A at 1.6 Å resolution along with extensive structural analyses did not indicate any evident difference with other characterized AA10 LPMOs at the catalytic interface, tempting us to suggest that these enzymes might also be active on xylan or that the ability to attack both crystalline and non-crystalline substrates involves yet obscure mechanisms of substrate recognition and binding. CONCLUSIONS: This work expands the spectrum of substrates recognized by AA10 family, opening a new perspective for the understanding of the synergistic effect of these enzymes with canonical glycoside hydrolases to deconstruct ligno(hemi)cellulosic biomass.
ABSTRACT
Citrus variegated chlorosis is a disease that attacks economically important citrus plantations and is caused by the plant-pathogenic bacterium Xylella fastidiosa. In this work, the structure of a small heat-shock protein from X. fastidiosa (XfsHSP17.9) is reported. The high-order structures of small heat-shock proteins from other organisms are arranged in the forms of double-disc, hollow-sphere or spherical assemblies. Unexpectedly, the structure reported here reveals a high-order architecture forming a nearly square cavity.
Subject(s)
Bacterial Proteins/chemistry , Heat-Shock Proteins, Small/chemistry , Heat-Shock Proteins/chemistry , Xylella/chemistry , Amino Acid Motifs , Bacterial Proteins/metabolism , Crystallography, X-Ray , Heat-Shock Proteins/metabolism , Heat-Shock Proteins, Small/metabolism , Models, Molecular , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Structural Homology, Protein , Xylella/metabolismABSTRACT
OBJECTIVES: To biochemically characterize an expansin-like X protein domain from Xanthomonas campestris (XcEXLX1) and to study its synergy with cellulases in cellulose depolymerization. RESULTS: The protein was purified using a combination of ion exchange and size exclusion chromatography rendering about 30 mg pure protein/l culture medium. Circular dichroism spectroscopy and small-angle X-ray scattering studies of XcEXLX1 reveal that it is a strongly disordered ß-sheet protein. Its low resolution envelope fits nicely the crystallographic structure of the homologous protein EXLX1 from Bacillus subtillis. Furthermore, we demonstrate that XcEXLX1 shows a synergistic, pH-dependent effect when combined with a commercial enzymatic preparation (Accellerase 1500), enhancing its hydrolytic activity on a cellulosic substrate. The strongest effect was observed in acid pHs with an increase in sugar release of up to 36 %. CONCLUSION: The synergistic effect arising from the action of the expansin-like protein was considerable in the presence of significantly larger amounts of the commercial enzymatic cocktail then previously observed (0.35 FPU of Accellerase 1500/g substrate).
