Dataset Information

Crystallographic structure of ChitA, a glycoside hydrolase family 19, plant class IV chitinase from Zea mays.

ABSTRACT: Maize ChitA chitinase is composed of a small, hevein-like domain attached to a carboxy-terminal chitinase domain. During fungal ear rot, the hevein-like domain is cleaved by secreted fungal proteases to produce truncated forms of ChitA. Here, we report a structural and biochemical characterization of truncated ChitA (ChitA ?N), which lacks the hevein-like domain. ChitA ?N and a mutant form (ChitA ?N-EQ) were expressed and purified; enzyme assays showed that ChitA ?N activity was comparable to the full-length enzyme. Mutation of Glu62 to Gln (ChitA ?N-EQ) abolished chitinase activity without disrupting substrate binding, demonstrating that Glu62 is directly involved in catalysis. A crystal structure of ChitA ?N-EQ provided strong support for key roles for Glu62, Arg177, and Glu165 in hydrolysis, and for Ser103 and Tyr106 in substrate binding. These findings demonstrate that the hevein-like domain is not needed for enzyme activity. Moreover, comparison of the crystal structure of this plant class IV chitinase with structures from larger class I and II enzymes suggest that class IV chitinases have evolved to accommodate shorter substrates.

SUBMITTER: Chaudet MM

PROVIDER: S-EPMC4005710 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Crystallographic structure of ChitA, a glycoside hydrolase family 19, plant class IV chitinase from Zea mays.

Chaudet Marcia M MM Naumann Todd A TA Price Neil P J NP Rose David R DR

Protein science : a publication of the Protein Society 20140310 5

Maize ChitA chitinase is composed of a small, hevein-like domain attached to a carboxy-terminal chitinase domain. During fungal ear rot, the hevein-like domain is cleaved by secreted fungal proteases to produce truncated forms of ChitA. Here, we report a structural and biochemical characterization of truncated ChitA (ChitA ΔN), which lacks the hevein-like domain. ChitA ΔN and a mutant form (ChitA ΔN-EQ) were expressed and purified; enzyme assays showed that ChitA ΔN activity was comparable to th ...[more]

PMID: 24616181

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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.

Dataset Information

Crystallographic structure of ChitA, a glycoside hydrolase family 19, plant class IV chitinase from Zea mays.

Publications

Crystallographic structure of ChitA, a glycoside hydrolase family 19, plant class IV chitinase from Zea mays.

Similar Datasets

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