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

ABSTRACT: 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.

SUBMITTER: Liberato MV

PROVIDER: S-EPMC4817029 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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

Liberato Marcelo V MV Silveira Rodrigo L RL Prates Érica T ÉT de Araujo Evandro A EA Pellegrini Vanessa O A VO Camilo Cesar M CM Kadowaki Marco A MA Neto Mario de O Mde O Popov Alexander A Skaf Munir S MS Polikarpov Igor I

Scientific reports 20160401

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 cat ...[more]

PMID: 27032335

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Project description:Chitinases degrade chitin into low molecular weight chitooligomers, which have a broad range of industrial, agricultural, and medical functions. Understanding the relationship between the diverse characteristics of chitinases and their functions is necessary for the improvement of functional enzymes that meet specific requirements. We report here a full crystallographic analysis of three complexes obtained from the chitinase Chit42 from Trichoderma harzianum, which represent different states along the enzymatic mechanism. The inactive double mutant D169A/E171A was submitted to soaking/crystallization experiments with hexa-N-acetyl-glucosamine (NAG6) or tetra-N-acetyl-glucosamine (NAG4), trapping the enzyme-substrate complex (Chit42-NAG6), the enzyme-products complex (Chit42-NAG4-NAG2) and a someway intermediate state. Structural comparison among the different complexes depicts the determinants defining the different subsites and revealed a previously unobserved dynamic on-off ligand binding process associated with a motion of its insertion domain, which might be accompanying the role or aromatics in processivity. An ensemble refinement performed to extract dynamic details from the diffraction data elucidates the implication of some highly flexible residues in the productive sliding of the substrate and the product release event. These positions were submitted to mutagenesis and the activity of the variants was investigated in the hydrolysis of NAG6, colloidal chitin and two chitosans with different polymerization and acetylation degree. All the changes affected the Chit42 hydrolytic activity therefore confirming the involvement of these positions in catalysis. Furthermore, we found the variants R295S and E316S improving the apparent catalytic efficiency of chitin and NAG6 and, together with E316A, enhancing the specific activity on chitosan. Therefore, our results provide novel insight into the molecular mechanisms underlying the hydrolysis of chitinous material by fungal chitinases, and suggest new targets to address engineering of these biotechnologically important enzymes.

Project description:Backgroundβ-mannanase can hydrolyze β-1,4 glycosidic bond of mannan by the manner of endoglycosidase to generate mannan-oligosaccharides. Currently, β-mannanase has been widely applied in food, medicine, textile, paper and petroleum exploitation industries. β-mannanase is widespread in various organisms, however, microorganisms are the main source of β-mannanases. Microbial β-mannanases display wider pH range, temperature range and better thermostability, acid and alkali resistance, and substrate specificity than those from animals and plants. Therefore microbial β-mannanases are highly valued by researchers. Recombinant bacteria constructed by gene engineering and modified by protein engineering have been widely applied to produce β-mannanase, which shows more advantages than traditional microbial fermentation in various aspects.ResultsA β-mannanase gene (Man1E), which encoded 731 amino acid residues, was cloned from Enterobacter aerogenes. Man1E was classified as Glycoside Hydrolase family 1. The bSiteFinder prediction showed that there were eight essential residues in the catalytic center of Man1E as Trp166, Trp168, Asn229, Glu230, Tyr281, Glu309, Trp341 and Lys374. The catalytic module and carbohydrate binding module (CBM) of Man1E were homologously modeled. Superposition analysis and molecular docking revealed the residues located in the catalytic module of Man1E and the CBM of Man1E. The recombinant enzyme was successfully expressed, purified, and detected about 82.5 kDa by SDS-PAGE. The optimal reaction condition was 55 °C and pH 6.5. The enzyme exhibited high stability below 60 °C, and in the range of pH 3.5-8.5. The β-mannanase activity was activated by low concentration of Co2+, Mn2+, Zn2+, Ba2+ and Ca2+. Man1E showed the highest affinity for Locust bean gum (LBG). The Km and Vmax values for LBG were 3.09 ± 0.16 mg/mL and 909.10 ± 3.85 μmol/(mL min), respectively.ConclusionsA new type of β-mannanase with high activity from E. aerogenes is heterologously expressed and characterized. The enzyme belongs to an unreported β-mannanase family (CH1 family). It displays good pH and temperature features and excellent catalysis capacity for LBG and KGM. This study lays the foundation for future application and molecular modification to improve its catalytic efficiency and substrate specificity.

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

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

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