Catalysis by Glomerella cingulata Cutinase Requires Conformational Cycling between the Active and Inactive States of Its Catalytic Triad

Nyon, Mun Peak; Rice, David W.; Berrisford, John M.; Hounslow, Andrea M.; Moir, Arthur J. G.; Huang, Huazhang; Nathan, Sheila; Mahadi, Nor Muhammad; Bakar, Farah Diba Abu; Craven, C. Jeremy

doi:10.1016/j.jmb.2008.10.050

Cited by 29 publications

(13 citation statements)

References 35 publications

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“…These three contrast with apoGcC where the loop carrying the catalytic histidine takes a quite different conformation causing the histidine to swing out from its expected position in the triad, reported to reflect importance of this movement in the catalytic cycle. 3 In the HiC complex with MEP covalently bound to S105, the density maps in addition clearly indicate H173 to have been ethylated, presumably a side product from the reaction with DNPP. More importantly, the loop carrying the ethylated H173 has swung out to break the catalytic triad and place it on the surface of the protein in a conformation reminiscent of that seen in apoGcC-but with a somewhat different final position.…”

Section: Structure Of Hicmentioning

confidence: 96%

“…A proposal for the significance of this histidine loop movement in relation to the mechanism has been extensively discussed. 3 The HiC-MEP complex structure…”

Section: Denaturation Of Hic Measured With Fluorescence Spectroscopymentioning

confidence: 99%

“…To date, the threedimensional (3D) structure has been reported for three cutinases, from Fusarium solani (FsC), 1 Glomerella cingulata (GcC), 2,3 and Aspergillus oryzae (AoC). 4 These enzymes are members of the a/b hydrolase superfamily with a central parallel bsheet and surrounding a-helices, and active site consisting of a catalytic triad of a serine, histidine, and aspartic acid.…”

Section: Introductionmentioning

confidence: 99%

“…As the SDS concentration is increased, HiC unfolds, leading to increased exposure of hydrophobic surfaces, and to the formation of dimers and possibly larger oligomers of HiC. (3). Further increasing the SDS concentration causes the oligomers to break up leading to micelle-like aggregates of SDS bound to the unfolded HiC.…”

Section: Introductionmentioning

confidence: 99%

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Thermodynamic and structural investigation of the specific SDS binding of humicola insolens cutinase

et al. 2014

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The interaction of lipolytic enzymes with anionic surfactants is of great interest with respect to industrially produced detergents. Here, we report the interaction of cutinase from the thermophilic fungus Humicola insolens with the anionic surfactant SDS, and show the enzyme specifically binds a single SDS molecule under nondenaturing concentrations. Protein interaction with SDS was investigated by NMR, ITC and molecular dynamics simulations. The NMR resonances of the protein were assigned, with large stretches of the protein molecule not showing any detectable resonances. SDS is shown to specifically interact with the loops surrounding the catalytic triad with medium affinity (K a % 10 5 M 21 ). The mode of binding is closely similar to that seen previously for binding of amphiphilic molecules and substrate analogues to cutinases, and hence SDS acts as a substrate mimic. In addition, the structure of the enzyme has been solved by X-ray crystallography in its apo form and after cocrystallization with diethyl p-nitrophenyl phosphate (DNPP) leading to a complex with monoethylphosphate (MEP) esterified to the catalytically active serine. TheAbbreviations: AA, all-atom; AoC, Aspergillus oryzae cutinase; AOT, sodium bis(2-ethylhexyl) sulfosuccinate; cmc, critical micelle concentration; DEP, diethylphosphate; DNPP, diethyl p-nitrophenyl phosphate; EDTA, ethylene diamine tetraacetate; FsC, Fusarium solani cutinase; GcC, Glomerella cingulata cutinase; HiC, Humicola insolens cutinase; HSQC, heteronuclear singlequantum coherence; IPTG, isopropyl b-D-1-thiogalactopyranoside; ITC, isothermal titration calorimetry; MD, molecular dynamics; MEP, monoethylphosphate; MWCO, molecular weight cut-off; PAGE, polyacrylamide gel electrophoresis; RMSD, root mean square deviation; RMSF, root mean square fluctuation; TES, N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid; SANS, small-angle neutron scattering; SDS, sodium dodecyl sulfate.Additional Supporting Information may be found in the online version of this article.An interactive view is available in the electronic version of the article. enzyme has the same fold as reported for other cutinases but, unexpectedly, esterification of the active site serine is accompanied by the ethylation of the active site histidine which flips out from its usual position in the triad.

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Section: Structure Of Hicmentioning

confidence: 96%

“…A proposal for the significance of this histidine loop movement in relation to the mechanism has been extensively discussed. 3 The HiC-MEP complex structure…”

Section: Denaturation Of Hic Measured With Fluorescence Spectroscopymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Thermodynamic and structural investigation of the specific SDS binding of humicola insolens cutinase

et al. 2014

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“…The crystal structures of two fungal cutinases from Fusarium solani f. sp. pisi (22) and Glomerella cingulata (27) have been determined. According to these structures, cutinase shares a common ␣/␤ hydrolase fold with lipase and esterase (28).…”

mentioning

confidence: 99%

Isolation of a Novel Cutinase Homolog with Polyethylene Terephthalate-Degrading Activity from Leaf-Branch Compost by Using a Metagenomic Approach

Sulaiman

Yamato

Kanaya

et al. 2012

Appl Environ Microbiol

513

385

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The gene encoding a cutinase homolog, LC-cutinase, was cloned from a fosmid library of a leaf-branch compost metagenome by functional screening using tributyrin agar plates. LC-cutinase shows the highest amino acid sequence identity of 59.7% to Thermomonospora curvata lipase. It also shows the 57.4% identity to Thermobifida fusca cutinase. When LCcutinase without a putative signal peptide was secreted to the periplasm of Escherichia coli cells with the assistance of the pelB leader sequence, more than 50% of the recombinant protein, termed LC-cutinase*, was excreted into the extracellular medium. It was purified and characterized. LC-cutinase* hydrolyzed various fatty acid monoesters with acyl chain lengths of 2 to 18, with a preference for short-chain substrates (C 4 substrate at most) most optimally at pH 8.5 and 50°C, but could not hydrolyze olive oil. It lost activity with half-lives of 40 min at 70°C and 7 min at 80°C. LC-cutinase* had an ability to degrade poly(-caprolactone) and polyethylene terephthalate (PET). The specific PET-degrading activity of LC-cutinase* was determined to be 12 mg/h/mg of enzyme (2.7 mg/h/kat of pNP-butyrate-degrading activity) at pH 8.0 and 50°C. This activity is higher than those of the bacterial and fungal cutinases reported thus far, suggesting that LC-cutinase* not only serves as a good model for understanding the molecular mechanism of PET-degrading enzyme but also is potentially applicable for surface modification and degradation of PET. C utinase (EC 3.1.1.74) is a lipolytic/esterolytic enzyme that hydrolyzes not only cutin, which is a major component of plant cuticle (38), but also water-soluble esters and insoluble triglycerides (12). It hydrolyzes these substrates to carboxylic acids and alcohols through the formation of an acyl enzyme intermediate, in which the active-site serine residue is acylated by the substrate. This serine residue is located within a GXSXG sequence motif and forms a catalytic triad with His and Asp. Cutinase has been found in both fungi and bacteria. The crystal structures of two fungal cutinases from Fusarium solani f. sp. pisi (22) and Glomerella cingulata (27) have been determined. According to these structures, cutinase shares a common ␣/␤ hydrolase fold with lipase and esterase (28). However, cutinase, like esterase, does not have a lid structure, which is responsible for interfacial activation of lipase (8). Therefore, cutinase does not show interfacial activation like esterase (14). Cutinase has recently received much attention because of its potential application for surface modification and degradation of aliphatic and aromatic polyesters (16), especially polyethylene terephthalate (PET), which is a synthetic aromatic polyester composed of terephthalic acid (TPA) and ethylene glycol (10,16,36,39). However, the number of cutinases, which have been studied regarding PET modification, is still limited, and this limitation may result in the delay of the research toward the practical use of cutinases. Therefore, isolation of a novel cutinase...

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2012

Fungi and Lignocellulosic Biomass

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Catalysis by Glomerella cingulata Cutinase Requires Conformational Cycling between the Active and Inactive States of Its Catalytic Triad

Cited by 29 publications

References 35 publications

Thermodynamic and structural investigation of the specific SDS binding of humicola insolens cutinase

Thermodynamic and structural investigation of the specific SDS binding of humicola insolens cutinase

Isolation of a Novel Cutinase Homolog with Polyethylene Terephthalate-Degrading Activity from Leaf-Branch Compost by Using a Metagenomic Approach

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