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Conformational plasticity and ligand binding of bacterial monoacylglycerol lipase.

ABSTRACT: Monoacylglycerol lipases (MGLs) play an important role in lipid catabolism across all kingdoms of life by catalyzing the release of free fatty acids from monoacylglycerols. The three-dimensional structures of human and a bacterial MGL were determined only recently as the first members of this lipase family. In addition to the ?/?-hydrolase core, they showed unexpected structural similarities even in the cap region. Nevertheless, the structural basis for substrate binding and conformational changes of MGLs is poorly understood. Here, we present a comprehensive study of five crystal structures of MGL from Bacillus sp. H257 in its free form and in complex with different substrate analogs and the natural substrate 1-lauroylglycerol. The occurrence of different conformations reveals a high degree of conformational plasticity of the cap region. We identify a specific residue, Ile-145, that might act as a gatekeeper restricting access to the binding site. Site-directed mutagenesis of Ile-145 leads to significantly reduced hydrolase activity. Bacterial MGLs in complex with 1-lauroylglycerol, myristoyl, palmitoyl, and stearoyl substrate analogs enable identification of the binding sites for the alkyl chain and the glycerol moiety of the natural ligand. They also provide snapshots of the hydrolytic reaction of a bacterial MGL at different stages. The alkyl chains are buried in a hydrophobic tunnel in an extended conformation. Binding of the glycerol moiety is mediated via Glu-156 and water molecules. Analysis of the structural features responsible for cap plasticity and the binding modes of the ligands suggests conservation of these features also in human MGL.

SUBMITTER: Rengachari S

PROVIDER: S-EPMC3829422 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Conformational plasticity and ligand binding of bacterial monoacylglycerol lipase.

Rengachari Srinivasan S Aschauer Philipp P Schittmayer Matthias M Mayer Nicole N Gruber Karl K Breinbauer Rolf R Birner-Gruenberger Ruth R Dreveny Ingrid I Oberer Monika M

The Journal of biological chemistry 20130906 43

Monoacylglycerol lipases (MGLs) play an important role in lipid catabolism across all kingdoms of life by catalyzing the release of free fatty acids from monoacylglycerols. The three-dimensional structures of human and a bacterial MGL were determined only recently as the first members of this lipase family. In addition to the α/β-hydrolase core, they showed unexpected structural similarities even in the cap region. Nevertheless, the structural basis for substrate binding and conformational chang ...[more]

PMID: 24014019

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Project description:BackgroundLipolytic enzymes of hyperthermophilic archaea generally prefer small carbon chain fatty acid esters (C2-C12) and are categorized as esterases. However, a few have shown activity with long-chain fatty acid esters, but none of them have been classified as a true lipase except a lipolytic enzyme AFL from Archaeglobus fulgidus. Thus, our main objective is to engineer an archaeal esterase into a true thermostable lipase for industrial applications. Lipases which hydrolyze long-chain fatty acid esters display an interfacial activation mediated by the lid domain which lies over active site and switches to open conformation at the oil-water interface. Lid domains modulate enzyme activities, substrate specificities, and stabilities which have been shown by protein engineering and mutational analyses. Here, we report engineering of an uncharacterized monoacylglycerol lipase (TON-LPL) from an archaeon Thermococcus onnurineus (strain NA1) into a triacylglycerol lipase (rc-TGL) by replacing its 61 N-terminus amino acid residues with 118 residues carrying lid domain of a thermophilic fungal lipase-Thermomyces lanuginosus (TLIP).ResultsTON-LPL and rc-TGL were cloned and overexpressed in E. coli, and the proteins were purified by Ni-NTA affinity chromatography for biochemical studies. Both enzymes were capable of hydrolyzing various monoglycerides and shared the same optimum pH of 7.0. However, rc-TGL showed a significant decrease of 10 °C in its optimum temperature (Topt). The far UV-CD spectrums were consistent with a well-folded α/β-hydrolase fold for both proteins, but gel filtration chromatography revealed a change in quaternary structure from trimer (TON-LPL) to monomer (rc-TGL). Seemingly, the difference in the oligomeric state of rc-TGL may be linked to a decrease in temperature optimum. Nonetheless, rc-TGL hydrolyzed triglycerides and castor oil, while TON-LPL was not active with these substrates.ConclusionsHere, we have confirmed the predicted esterase activity of TON-LPL and also performed the lid engineering on TON-LPL which effectively expanded its substrate specificity from monoglycerides to triglycerides. This approach provides a way to engineer other hyperthermophilic esterases into industrially suitable lipases by employing N-terminal domain replacement. The immobilized preparation of rc-TGL has shown significant activity with castor oil and has a potential application in castor oil biorefinery to obtain value-added chemicals.

Dataset Information

Conformational plasticity and ligand binding of bacterial monoacylglycerol lipase.

Publications

Conformational plasticity and ligand binding of bacterial monoacylglycerol lipase.

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