Exploring the Molecular Basis for Substrate Affinity and Structural Stability in Bacterial GH39 ß-Xylosidases.

The glycoside hydrolase family 39 (GH39) is a functionally expanding family with limited understanding about the molecular basis for substrate specificity and extremophilicity. In this work, we demonstrate the key role of the positive-subsite region in modulating substrate affinity and how the lack of a C-terminal extension impacts on oligomerization and structural stability of some GH39 members. The crystallographic and SAXS structures of a new GH39 member from the phytopathogen Xanthomonas citri support the importance of an extended C-terminal to promote oligomerization as a molecular strategy to enhance thermal stability. Comparative structural analysis along with site-directed mutagenesis showed that two residues located at the positive-subsite region, Lys166 and Asp167, are critical to substrate affinity and catalytic performance, by inducing local changes in the active site for substrate binding. These findings expand the molecular understanding of the mechanisms involved in substrate recognition and structural stability of the GH39 family, which might be instrumental for biological insights, rational enzyme engineering and utilization in biorefineries.

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

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.

Structural basis of exo-ß-mannanase activity in the GH2 family.

The classical microbial strategy for depolymerization of ß-mannan polysaccharides involves the synergistic action of at least two enzymes, endo-1,4-ß-mannanases and ß-mannosidases. In this work, we describe the first exo-ß-mannanase from the GH2 family, isolated from Xanthomonas axonopodis pv. citri (XacMan2A), which can efficiently hydrolyze both manno-oligosaccharides and ß-mannan into mannose. It represents a valuable process simplification in the microbial carbon uptake that could be of potential industrial interest. Biochemical assays revealed a progressive increase in the hydrolysis rates from mannobiose to mannohexaose, which distinguishes XacMan2A from the known GH2 ß-mannosidases. Crystallographic analysis indicates that the active-site topology of XacMan2A underwent profound structural changes at the positive-subsite region, by the removal of the physical barrier canonically observed in GH2 ß-mannosidases, generating a more open and accessible active site with additional productive positive subsites. Besides that, XacMan2A contains two residue substitutions in relation to typical GH2 ß-mannosidases, Gly439 and Gly556, which alter the active site volume and are essential to its mode of action. Interestingly, the only other mechanistically characterized mannose-releasing exo-ß-mannanase so far is from the GH5 family, and its mode of action was attributed to the emergence of a blocking loop at the negative-subsite region of a cleft-like active site, whereas in XacMan2A, the same activity can be explained by the removal of steric barriers at the positive-subsite region in an originally pocket-like active site. Therefore, the GH2 exo-ß-mannanase represents a distinct molecular route to this rare activity, expanding our knowledge about functional convergence mechanisms in carbohydrate-active enzymes.

Structural basis for xyloglucan specificity and α-d-Xylp(1 → 6)-D-Glcp recognition at the -1 subsite within the GH5 family.

GH5 is one of the largest glycoside hydrolase families, comprising at least 20 distinct activities within a common structural scaffold. However, the molecular basis for the functional differentiation among GH5 members is still not fully understood, principally for xyloglucan specificity. In this work, we elucidated the crystal structures of two novel GH5 xyloglucanases (XEGs) retrieved from a rumen microflora metagenomic library, in the native state and in complex with xyloglucan-derived oligosaccharides. These results provided insights into the structural determinants that differentiate GH5 XEGs from parental cellulases and a new mode of action within the GH5 family related to structural adaptations in the -1 subsite. The oligosaccharide found in the XEG5A complex, permitted the mapping, for the first time, of the positive subsites of a GH5 XEG, revealing the importance of the pocket-like topology of the +1 subsite in conferring the ability of some GH5 enzymes to attack xyloglucan. Complementarily, the XEG5B complex covered the negative subsites, completing the subsite mapping of GH5 XEGs at high resolution. Interestingly, XEG5B is, to date, the only GH5 member able to cleave XXXG into XX and XG, and in the light of these results, we propose that a modification in the -1 subsite enables the accommodation of a xylosyl side chain at this position. The stereochemical compatibility of the -1 subsite with a xylosyl moiety was also reported for other structurally nonrelated XEGs belonging to the GH74 family, indicating it to be an essential attribute for this mode of action.

Development and biotechnological application of a novel endoxylanase family GH10 identified from sugarcane soil metagenome.

Metagenomics has been widely employed for discovery of new enzymes and pathways to conversion of lignocellulosic biomass to fuels and chemicals. In this context, the present study reports the isolation, recombinant expression, biochemical and structural characterization of a novel endoxylanase family GH10 (SCXyl) identified from sugarcane soil metagenome. The recombinant SCXyl was highly active against xylan from beechwood and showed optimal enzyme activity at pH 6,0 and 45°C. The crystal structure was solved at 2.75 Å resolution, revealing the classical (ß/α)8-barrel fold with a conserved active-site pocket and an inherent flexibility of the Trp281-Arg291 loop that can adopt distinct conformational states depending on substrate binding. The capillary electrophoresis analysis of degradation products evidenced that the enzyme displays unusual capacity to degrade small xylooligosaccharides, such as xylotriose, which is consistent to the hydrophobic contacts at the +1 subsite and low-binding energies of subsites that are distant from the site of hydrolysis. The main reaction products from xylan polymers and phosphoric acid-pretreated sugarcane bagasse (PASB) were xylooligosaccharides, but, after a longer incubation time, xylobiose and xylose were also formed. Moreover, the use of SCXyl as pre-treatment step of PASB, prior to the addition of commercial cellulolytic cocktail, significantly enhanced the saccharification process. All these characteristics demonstrate the advantageous application of this enzyme in several biotechnological processes in food and feed industry and also in the enzymatic pretreatment of biomass for feedstock and ethanol production.

Molecular insights into substrate specificity and thermal stability of a bacterial GH5-CBM27 endo-1,4-ß-D-mannanase.

The breakdown of ß-1,4-mannoside linkages in a variety of mannan-containing polysaccharides is of great importance in industrial processes such as kraft pulp delignification, food processing and production of second-generation biofuels, which puts a premium on studies regarding the prospection and engineering of ß-mannanases. In this work, a two-domain ß-mannanase from Thermotoga petrophila that encompasses a GH5 catalytic domain with a C-terminal CBM27 accessory domain, was functionally and structurally characterized. Kinetic and thermal denaturation experiments showed that the CBM27 domain provided thermo-protection to the catalytic domain, while no contribution on enzymatic activity was observed. The structure of the catalytic domain determined by SIRAS revealed a canonical (α/ß)(8)-barrel scaffold surrounded by loops and short helices that form the catalytic interface. Several structurally related ligand molecules interacting with TpMan were solved at high-resolution and resulted in a wide-range representation of the subsites forming the active-site cleft with residues W134, E198, R200, E235, H283 and W284 directly involved in glucose binding.

Functional and biophysical characterization of a hyperthermostable GH51 α-L-arabinofuranosidase from Thermotoga petrophila.

A hyperthermostable glycoside hydrolase family 51 (GH51) α-L-arabinofuranosidase from Thermotoga petrophila RKU-1 (TpAraF) was cloned, overexpressed, purified and characterized. The recombinant enzyme had optimum activity at pH 6.0 and 70°C with linear α-1,5-linked arabinoheptaose as substrate. The substrate cleavage pattern monitored by capillary zone electrophoresis showed that TpAraF is a classical exo-acting enzyme producing arabinose as its end-product. Far-UV circular dichroism analysis displayed a typical spectrum of α/ß barrel proteins analogously observed for other GH51 α-L-arabinofuranosidases. Moreover, TpAraF was crystallized in two crystalline forms, which can be used to determine its crystallographic structure.

Crystallization and preliminary X-ray diffraction analysis of Q4DV70 from Trypanosoma cruzi, a hypothetical protein with a putative thioredoxin domain.

Q4DV70 is annotated in the Trypanosoma cruzi CL Brener genome as a hypothetical protein with a predicted thioredoxin-like fold, although the catalytic cysteine residues that are conserved in typical oxidoreductases are replaced by serine residues. Gene-expression analysis indicates that this protein is differentially expressed during the T. cruzi life cycle, suggesting that it plays an important role during T. cruzi development. The gene coding for Q4DV70 was cloned and the protein was overexpressed in Escherichia coli with an N-terminal His tag. Purification of Q4DV70 was carried out by affinity and size-exclusion chromatography and the His tag was removed by TEV protease digestion. Crystals of Q4DV70 were grown using the sitting-drop vapour-diffusion method. A diffraction data set was collected to 1.50 A resolution from a single crystal grown in 25% PEG 1500, 200 mM sodium thiocyanate pH 6.9, 10 mM phenol and 10% ethylene glycol. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 35.04, b = 50.32, c = 61.18 A. The Q4DV70 structure was solved by molecular replacement using protein disulfide isomerase from yeast (PDB code 2b5e) as a search model. Initial refinement of the model indicated that the solution was correct. These data are being used for refinement of the model of Q4DV70.

Purification, crystallization and preliminary crystallographic studies of SPCI-chymotrypsin complex at 2.8 A resolution.

A binary complex of the Schizolobium parahyba chymotrypsin inhibitor (SPCI) with chymotrypsin was purified by size-exclusion chromatography and crystallized by the sitting-drop vapour-diffusion method with 100 mM MES-NaOH pH 5.5, 20%(w/v) PEG 6000, 200 mM LiCl as precipitant and 200 mM nondetergent sulfobetaine molecular weight 201 Da (NDSB-201) as an additive. SPCI is a small protein with 180 amino-acid residues isolated from S. parahyba seeds and is able to inhibit chymotrypsin at a 1:1 molar ratio by forming a stable complex. X-ray data were collected to 2.8 A resolution from a single crystal of the SPCI-chymotrypsin binary complex under cryogenic conditions. The crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 45.28, b = 64.57, c = 169.23 A, and the R(merge) is 0.122 for 11 254 unique reflections. A molecular-replacement solution was found using the preliminary crystal structure of SPCI and the structure of chymotrypsin (PDB code 4cha) independently as search models.