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

Abstract

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.

Screening of commercial enzymes for poly(ethylene terephthalate) (PET) hydrolysis and synergy studies on different substrate sources

“…Interestingly, cutinases usually show lower molecular weight (20-30 kDa) than lipases [19], in accordance to the results we found in this present study, with up to 70 kDa bands (from RnL) (Table 1S). For this study, it was prioritized a condition in which all enzymes could be active and stable, more than an ideal condition of PET availability, such as near to its glass transition temperature.…”

Spatially confined lignin nanospheres for biocatalytic ester synthesis in aqueous media

“…Hydrolases act as gatekeepers that enhance the selective permeation of butanol and butyric acid. Hydrophobic residues are shown in red color for the HiC molecules 46 . d Diffusion of the produced butyl butyrate into the organic phase is driven by agitation that sequentially brings the biocatalyst beads to the solvent interface …”

High-level expression and characterization of a novel cutinase from Malbranchea cinnamomea suitable for butyl butyrate production

“…In general, enzymes from thermophilic microorganisms display relatively high optimal temperature and good thermostability compared to those from mesophilic microorganisms. However, there are few reports on cutinases from thermophilic fungi, except for Humicola insolens [ 10 ] and Thielavia terrestris [ 11 , 12 ]. Although many cutinase genes have been cloned and expressed, few reports are available on bioreactor systems for large-scale production [ 8 , 13 – 16 ].…”

“…The crystal structure of the cutinase from Fusarium solani pisi was firstly determined and thoroughly studied [ 20 , 21 ]. Structures of fungal cutinases from Aspergillus oryzae [ 22 ], Fusarium oxysporum [ 23 ], Glomerella cingulata [ 24 ], H. insolens [ 10 ], and Trichoderma reesei [ 25 ], and two bacterial cutinases from Thermobifida alba [ 26 ] and T. fusca [ 27 ] have been solved. Structural studies of cutinases can help further understand their action modes and obtain more useful information for the redesign of enzymes to fulfill industrial application requirements.…”