Crystallization and preliminary X-ray diffraction analysis of a new xyloglucanase from Xanthomonas campestris pv. campestris

Xyloglucanases (Xghs) are important enzymes involved in xyloglucan modification and degradation. Xanthomonas campestris pv. campestris (Xcc) is a phytopathogenic bacterium which produces a large number of glycosyl hydrolases (GH), but has only one family 74 GH (Xcc-Xgh). This enzyme was overexpressed in Escherichia coli, purified and crystallized. Diffraction data sets were collected for the native enzyme and its complex with glucose to maximum resolutions of 2.0 and 2.1 Å respectively. The data were indexed in a hexagonal crystal system with unit-cell parameters a = b = 153.4, c = 84.9 Å. As indicated by molecular-replacement solution, the crystals belonged to space group P61.FAPESP (08/56255-9, 07/08706-9, 10/52362-5, 09/05349-6)CAPESCNPq / INCT do Bioetanol (471834/2009-2, 301981/2011-6, 550931/2011-2

Effect of dynamic high pressure on functional and structural properties of bovine serum albumin

Dynamic high pressure (DHP) has been investigated as an innovative suitable method to induce protein modifications. This work evaluated the effect of DHP (up to three passes at 100, 150 and 200 MPa, with an inlet temperature of 20 °C) on functional and structural properties of bovine serum albumin (BSA). Results indicated that DHP process applied up to an energy limit of 100 MPa increased the protein foaming capacity (FC) (p < 0.05 - increase up to 63% after 1 pass at 100 MPa) and the utilization of multiple passes at high pressure promoted a reduction in this property (p < 0.05 - reduction up to 31.6% after 3 passes at 200 MPa). Similar results were observed for sulfhydryl group, indicating an influence of free thiol groups on FC. Complementarily, DHP process promoted an increase of proteins particles size, suggesting a new rearrangement of their conformational structure. DHP did not affect tryptophan microenvironment in BSA; however, this process induced the rearrangement of secondary structure elements. In the first cycle, the pressure increase resulted in a loss of secondary structure, while in the second and third cycles the DHP process resulted in the gain of secondary structure elements. These results indicated that the second and third passes triggered a molecular rearrangement of the protein structure, giving rise to a novel and more stable conformational state. This conclusion was also supported by thermal unfolding studies (melting temperature reduction from 67.5 to 54.6 °C after 1 pass at 200 MPa), in which the additional cycles of DHP caused the occurrence of an initial denaturation at high temperatures, compared to the first cycle

Structural studies of the Trypanosoma cruzi Old Yellow Enzyme: insights into enzyme dynamics and specificity

The flavoprotein old yellowenzyme of Trypanosoma cruzi (TcOYE) is an oxidoreductase that usesNAD(P)H as cofactor. This enzyme is clinically relevant due to its role in the action mechanismof some trypanocidal drugs used in the treatment of Chagas' disease by producing reactive oxygen species. In thiswork, the recombinant enzyme TcOYE was produced and collectively, X-ray crystallography, small angle X-ray scattering, analytical ultracentrifugation and molecular dynamics provided a detailed description of its structure, specificity and hydrodynamic behavior. The crystallographic structure at 1.27 Å showed a classical (α/β)8 fold with the FMN prosthetic group buried at the positively-charged active-site cleft. In solution, TcOYE behaved as a globular monomer, but it exhibited a molecular envelope larger than that observed in the crystal structure, suggesting intrinsic protein\ud flexibility. Moreover, the binding mode of β-lapachone, a trypanocidal agent, and other naphthoquinones was investigated by molecular docking and dynamics suggesting that their binding to TcOYE are stabilized mainly by interactions with the isoalloxazine ring from FMN and residues from the active-site pocket.FAPESP (07/05001-4, 11/23110-0)CNP

The mechanism by which a distinguishing arabinofuranosidase can cope with internal di-substitutions in arabinoxylans

Abstract Background Arabinoxylan is an abundant polysaccharide in industrially relevant biomasses such as sugarcane, corn stover and grasses. However, the arabinofuranosyl di-substitutions that decorate the xylan backbone are recalcitrant to most known arabinofuranosidases (Abfs). Results In this work, we identified a novel GH51 Abf (XacAbf51) that forms trimers in solution and can cope efficiently with both mono- and di-substitutions at terminal or internal xylopyranosyl units of arabinoxylan. Using mass spectrometry, the kinetic parameters of the hydrolysis of 33-α-l-arabinofuranosyl-xylotetraose and 23,33-di-α-l-arabinofuranosyl-xylotetraose by XacAbf51 were determined, demonstrating the capacity of this enzyme to cleave arabinofuranosyl linkages of internal mono- and di-substituted xylopyranosyl units. Complementation studies of fungal enzyme cocktails with XacAbf51 revealed an increase of up to 20% in the release of reducing sugars from pretreated sugarcane bagasse, showing the biotechnological potential of a generalist GH51 in biomass saccharification. To elucidate the structural basis for the recognition of internal di-substitutions, the crystal structure of XacAbf51 was determined unveiling the existence of a pocket strategically arranged near to the − 1 subsite that can accommodate a second arabinofuranosyl decoration, a feature not described for any other GH51 Abf structurally characterized so far. Conclusions In summary, this study reports the first kinetic characterization of internal di-substitution release by a GH51 Abf, provides the structural basis for this activity and reveals a promising candidate for industrial processes involving plant cell wall depolymerization