Marco Antonio Seiki Kadowaki

Aspergillus niger β-glucosidase has a cellulase-like tadpole molecular shape: insights into glycoside hydrolase family 3 (GH3) β-glucosidase structure and function.

Background: β-Glucosidase completes cellulose enzymatic hydrolysis by releasing glucose from cellobiose. Results: SAXS experiments revealed that Aspergillus niger β-glucosidase has a cellulase-like tadpole molecular shape, uncommon to enzymes that act on the soluble substrates. Conclusion: We show that AnBgl1 N- and C-terminal domains are linked by a long extended linker. Significance: Understanding AnBgl1 architecture is useful for comprehension of the enzyme-cell wall interaction and the process of biomass saccharification. Aspergillus niger is known to secrete large amounts of β-glucosidases, which have a variety of biotechnological and industrial applications. Here, we purified an A. niger β-glucosidase (AnBgl1) and conducted its biochemical and biophysical analyses. Purified enzyme with an apparent molecular mass of 116 kDa forms monomers in solution as judged by native gel electrophoresis and has a pI value of 4.55, as found for most of the fungi of β-glucosidases. Surprisingly, the small angle x-ray experiments reveal that AnBgl1 has a tadpole-like structure, with the N-terminal catalytic domain and C-terminal fibronectin III-like domain (FnIII) connected by the long linker peptide (∼100 amino acid residues) in an extended conformation. This molecular organization resembles the one adopted by other cellulases (such as cellobiohydrolases, for example) that frequently contain a catalytic domain linked to the cellulose-binding module that mediates their binding to insoluble and polymeric cellulose. The reasons why AnBgl1, which acts on the small soluble substrates, has a tadpole molecular shape are not entirely clear. However, our enzyme pulldown assays with different polymeric substrates suggest that AnBgl1 has little or no capacity to bind to and to adsorb cellulose, xylan, and starch, but it has high affinity to lignin. Molecular dynamics simulations suggested that clusters of residues located in the C-terminal FnIII domain interact strongly with lignin fragments. The simulations showed that numerous arginine residues scattered throughout the FnIII surface play an important role in the interaction with lignin by means of cation-π stacking with the lignin aromatic rings. These results indicate that the C-terminal FnIII domain could be operational for immobilization of the enzyme on the cell wall and for the prevention of unproductive binding of cellulase to the biomass lignin.

Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism

Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms. The Ig-like module is pivoting the packing and unpacking motions of CBM46 relative to CD in the assembly of the binding subsite. This is the first example of a multidomain GH relying on large amplitude motions of the CBM46 for assembly of the catalytically competent form of the enzyme.

Biochemical characterization and low-resolution SAXS structure of an exo-polygalacturonase from Bacillus licheniformis

Among the structural polymers present in the plant cell wall, pectin is the main component of the middle lamella. This heterogeneous polysaccharide has an α-1,4 galacturonic acid backbone, which can be broken by the enzymatic action of pectinases, such as exo-polygalacturonases, that sequentially cleave pectin from the non-reducing ends, releasing mono or di-galacturonic acid residues. Constant demand for pectinases that better suit industrial requirements has motivated identification and characterization of novel enzymes from diverse sources. Bacillus licheniformis has been used as an important source for bioprospection of several industrial biomolecules, such as surfactants and enzymes, including pectate lyases. Here we cloned, expressed, purified, and biochemically and structurally characterized an exo-polygalacturonase from B. licheniformis (BlExoPG). Its low-resolution molecular envelope was derived from experimental small-angle scattering data (SAXS). Our experimental data revealed that BlExoPG is a monomeric enzyme with optimum pH at 6.5 and optimal temperature of approximately 60°C, at which it has considerable stability over the broad pH range from 5 to 10. After incubation of the enzyme for 30min at pH ranging from 5 to 10, no significant loss of the original enzyme activity was observed. Furthermore, the enzyme maintained residual activity of greater than 80% at 50°C after 15h of incubation. BlExoPG is more active against polygalacturonic acid as compared to methylated pectin, liberating mono galacturonic acid as a unique product. Its enzymatic parameters are Vmax=4.18μM.s-1,Km=3.25mgmL-1 and kcat=2.58s-1.

Crystal structure of β1→6‐galactosidase from Bifidobacterium bifidum S17: trimeric architecture, molecular determinants of the enzymatic activity and its inhibition by α‐galactose

In a search for better comprehension of β‐galactosidase function and specificity, we solved the crystal structures of the GH42 β‐galactosidase BbgII from Bifidobacterium bifidum S17, a well‐adapted probiotic microorganism from the human digestive tract, and its complex with d‐α‐galactose. BbgII is a three‐domain molecule that forms barrel‐shaped trimers in solution. BbgII interactions with d‐α‐galactose, a competitive inhibitor, showed a number of residues that are involved in the coordination of ligands. A combination of site‐directed mutagenesis of these amino acid residues with enzymatic activity measurements confirmed that Glu161 and Glu320 are fundamental for catalysis and their substitution by alanines led to catalytically inactive mutants. Mutation Asn160Ala resulted in a two orders of magnitude decrease of the enzyme kcat without significant modification in its Km, whereas mutations Tyr289Phe and His371Phe simultaneously decreased kcat and increased Km values. Enzymatic activity of Glu368Ala mutant was too low to be detected. Our docking and molecular dynamics simulations showed that the enzyme recognizes and tightly binds substrates with β1→6 and β1→3 bonds, while binding of the substrates with β1→4 linkages is less favorable.

Structure, computational and biochemical analysis of PcCel45A endoglucanase from Phanerochaete chrysosporium and catalytic mechanisms of GH45 subfamily C members

The glycoside hydrolase family 45 (GH45) of carbohydrate modifying enzymes is mostly comprised of β-1,4-endoglucanases. Significant diversity between the GH45 members has prompted the division of this family into three subfamilies: A, B and C, which may differ in terms of the mechanism, general architecture, substrate binding and cleavage. Here, we use a combination of X-ray crystallography, bioinformatics, enzymatic assays, molecular dynamics simulations and site-directed mutagenesis experiments to characterize the structure, substrate binding and enzymatic specificity of the GH45 subfamily C endoglucanase from Phanerochaete chrysosporium (PcCel45A). We investigated the role played by different residues in the binding of the enzyme to cellulose oligomers of different lengths and examined the structural characteristics and dynamics of PcCel45A that make subfamily C so dissimilar to other members of the GH45 family. Due to the structural similarity shared between PcCel45A and domain I of expansins, comparative analysis of their substrate binding was also carried out. Our bioinformatics sequence analyses revealed that the hydrolysis mechanisms in GH45 subfamily C is not restricted to use of the imidic asparagine as a general base in the “Newton’s cradle” catalytic mechanism recently proposed for this subfamily.

Low-resolution envelope, biophysical analysis and biochemical characterization of a short-chain specific and halotolerant carboxylesterase from Bacillus licheniformis

Esterases are widely applied in industrial processes due to their versatility, regio- and enantioselectivity, lack of cofactors and stability in organic solvents. Bacillus licheniformis, a microorganism frequently used in industrial and biotechnological applications such as dairy, baking, beverage, pulp and paper, detergent and cosmetics production, organic synthesis and waste management, is a promising source of esterases. Here we describe the biochemical and biophysical characterization of B. licheniformis carboxylesterase BlEst1 and its SAXS-derived molecular envelope. BlEst1 has optimal hydrolytic activity against p‑nitrophenyl acetate at pH 7.0 and 40 °C. Furthermore, BlEst1 is stable in different organic solvents such as methanol, isopropanol and butanol. The BlEst1 homology model reveals a typical α/β hydrolase core with an adjacent auxiliary domain, snuggly fitting the experimental low-resolution SAXS molecular envelope. Moreover, BlEst1 maintained considerable part of its activity in the presence of up to 5 M NaCl and its thermal stability was significantly enhanced by the presence of salt, revealing its halotolerant character. The ability to work under harsh conditions makes BlEst1 an interesting candidate for industrial applications.

Biochemical and structural insights into a thermostable cellobiohydrolase from Myceliophthora thermophila

Cellobiohydrolases hydrolyze cellulose, a linear polymer with glucose monomers linked exclusively by β‐1,4 glycosidic linkages. The widespread hydrogen bonding network tethers individual cellulose polymers forming crystalline cellulose, which prevent the access of hydrolytic enzymes and water molecules. The most abundant enzyme secreted by Myceliophthora thermophila M77 in response to the presence of biomass is the cellobiohydrolase MtCel7A, which is composed by a GH7‐catalytic domain (CD), a linker, and a CBM1‐type carbohydrate‐binding module. GH7 cellobiohydrolases have been studied before, and structural models have been proposed. However, currently available GH7 crystal structures only define separate catalytic domains and/or cellulose‐binding modules and do not include the full‐length structures that are involved in shaping the catalytic mode of operation. In this study, we determined the 3D structure of catalytic domain using X‐ray crystallography and retrieved the full‐length enzyme envelope via small‐angle X‐ray scattering (SAXS) technique. The SAXS data reveal a tadpole‐like molecular shape with a rigid linker connecting the CD and CBM. Our biochemical studies show that MtCel7A has higher catalytic efficiency and thermostability as well as lower processivity when compared to the well‐studied TrCel7A from Trichoderma reesei. Based on a comparison of the crystallographic structures of CDs and their molecular dynamic simulations, we demonstrate that MtCel7A has considerably higher flexibility than TrCel7A. In particular, loops that cover the active site are more flexible and undergo higher conformational fluctuations, which might account for decreased processivity and enhanced enzymatic efficiency. Our statistical coupling analysis suggests co‐evolution of amino acid clusters comprising the catalytic site of MtCel7A, which correlate with the steps in the catalytic cycle of the enzyme.

Biochemical characterization, low-resolution SAXS structure and an enzymatic cleavage pattern of Bl Cel48 from Bacillus licheniformis

Economic sustainability of modern biochemical technologies for plant cell wall transformations in renewable fuels, green chemicals, and sustainable materials is considerably impacted by the elevated cost of enzymes. Therefore, there is a significant drive toward discovery and characterization of novel carbohydrate-active enzymes. Here, the BlCel48 cellulase from Bacillus licheniformis, a glycoside hydrolase family 48 member (GH48), was functionally and biochemically characterized. The enzyme is catalytically stable in a broad range of temperatures and pH conditions with its enzymatic activity at pH5.0 and 60°C. BlCel48 exhibits high hydrolytic activity against phosphoric acid swollen cellulose (PASC) and bacterial cellulose (BC) and significantly lower activity against carboxymethylcellulose (CMC). BlCel48 releases predominantly cellobiose, and also small amounts of cellotriose and cellotetraose as products from PASC hydrolysis. Small-angle X-ray scattering (SAXS) data analysis revealed a globular molecular shape and monomeric state of the enzyme in solution. Its molecular mass estimated based on SAXS data is ~77.2kDa. BlCel48 has an (αα)6-helix barrel-fold, characteristic of GH48 members. Comparative analyses of homologous sequences and structures reveal the existence of two distinct loops in BlCel48 that were not present in other structurally characterized GH48 enzymes which could have importance for the enzyme activity and specificity.

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 P6(1).