The Role of Residues 103, 104, and 278 in the Activity of SMG1 Lipase from Malassezia globosa: A Site-Directed Mutagenesis Study
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The SMG1 lipase from Malassezia globosa is a newly found mono- and diacylglycerol (DAG) lipase that has a unique lid in the loop conformation that differs from the common alpha-helix lid. In the present study, we characterized the contribution of three residues, L103 and F104 in the lid and F278 in the rim of the binding site groove, on the function of SMG1 lipase. Sitedirected mutagenesis was conducted at these sites, and each of the mutants was expressed in the yeast Pichia pastoris, purified, and characterized for their activity toward DAG and pnitrophenol (pNP) ester. Compared with wild-type SMG1, F278A retained approximately 78% of its activity toward DAG, but only 11% activity toward pNP octanoate (pNP-C8). L103G increased its activity on pNP-C8 by approximately 2-fold, whereas F104G showed an approximate 40% decrease in pNP-C8 activity, and they both showed decreased activity on the DAG emulsion. The deletion of 103-104 retained approximately 30% of its activity toward the DAG emulsion, with an almost complete loss of pNP-C8 activity. The deletion of 103-104 showed a weaker penetration ability to a soybean phosphocholine monolayer than wild-type SMG1. Based on the modulation of the specificity and activity observed, a pNP-C8 binding model for the ester (pNP-C8, N102, and F278 form a flexible bridge) and a specific lipidanchoring mechanism for DAG (L103 and F104 serve as “anchors” to the lipid interface) were proposed.
Abstract
The SMG1 lipase from Malassezia globosa is a newly found mono- and diacylglycerol (DAG) lipase that has a unique lid in the loop conformation that differs from the common alpha-helix lid. In the present study, we characterized the contribution of three residues, L103 and F104 in the lid and F278 in the rim of the binding site groove, on the function of SMG1 lipase. Sitedirected mutagenesis was conducted at these sites, and each of the mutants was expressed in the yeast Pichia pastoris, purified, and characterized for their activity toward DAG and pnitrophenol (pNP) ester. Compared with wild-type SMG1, F278A retained approximately 78% of its activity toward DAG, but only 11% activity toward pNP octanoate (pNP-C8). L103G increased its activity on pNP-C8 by approximately 2-fold, whereas F104G showed an approximate 40% decrease in pNP-C8 activity, and they both showed decreased activity on the DAG emulsion. The deletion of 103-104 retained approximately 30% of its activity toward the DAG emulsion, with an almost complete loss of pNP-C8 activity. The deletion of 103-104 showed a weaker penetration ability to a soybean phosphocholine monolayer than wild-type SMG1. Based on the modulation of the specificity and activity observed, a pNP-C8 binding model for the ester (pNP-C8, N102, and F278 form a flexible bridge) and a specific lipidanchoring mechanism for DAG (L103 and F104 serve as “anchors” to the lipid interface) were proposed.
참고문헌 (27)
- Akoh CC, "Enzymatic approach to biodiesel production", J. Agric. Food Chem, 55, pp.8995~9005 (2007)
- Ben Salah A, "Kinetic studies of Rhizopus oryzae lipase using monomolecular film technique", Biochimie, 83, pp.463~469 (2001)
- Brady L, "A serine protease triad forms the catalytic centre of a triacylglycerol lipase", Nature, 343, pp.767~770 (1990)
- Brocca S, "Sequence of the lid affects activity and specificity of Candida rugosa lipase isoenzymes", Protein Sci, 12, pp.2312~2319 (2003)
- Brzozowski AM, "Structural origins of the interfacial activation in Thermomyces (Humicola)lanuginosa lipase", Biochemistry (Mosc.), 39, pp.15071~15082 (2000)
- Cao M, "Homogenization and lipase treatment of milk and resulting methyl ketone generation in blue cheese", J. Agric. Food Chem, 62, pp.5726~5733 (2014)
- DeAngelis YM, "Isolation and expression of a Malassezia globosa lipase gene, LIP1", J. Invest. Dermatol, 127, pp.2138~2146 (2007)
- Dugi KA, "Human hepatic and lipoprotein lipase: the loop covering the catalytic site mediates lipase substrate specificity", J. Biol. Chem, 270, pp.25396~25401 (1995)
- Emsley P, "Features and development of Coot", Acta Crystallogr. D, 66, pp.486~501 (2010)
- Gao CL, "Site-directed mutagenesis studies of the aromatic residues at the active site of a lipase from Malassezia globosa", Biochimie, 102, pp.29~36 (2014)
- Holmquist M, "Trp89 in the lid of Humicola lanuginosa lipase is important for efficient hydrolysis of tributyrin", Lipids, 29, pp.599~603 (1994)
- Lan D, "Conversion of a mono- and diacylglycerol lipase into a triacylglycerol lipase by protein engineering", ChemBioChem, 16, pp.1431~1434 (2015)
- Martinelle M, "On the interfacial activation of Candida antarctica lipase A and B as compared with Humicola lanuginosa lipase", Biochim. Biophys. Acta, 1258, pp.272~276 (1995)
- Noble ME, "The crystal structure of triacylglycerol lipase from Pseudomonas glumae reveals a partially redundant catalytic aspartate", FEBS Lett, 331, pp.123~128 (1993)
- Nomura DK, "Monoacylglycerol lipase regulates a fatty acid network that promotes cancer pathogenesis", Cell, 140, pp.49~61 (2010)
- Pan XX, "Efficient display of active Geotrichum sp. lipase on Pichia pastoris cell wall and its application as a whole-cell biocatalyst to enrich EPA and DHA in fish oil", J. Agric. Food Chem, 60, pp.9673~9679 (2012)
- Qin XL, "Lipase-catalyzed incorporation of different fatty acids into tripalmitin-enriched triacylglycerols: effect of reaction parameters", J. Agric. Food Chem, 60, pp.2377~2384 (2012)
- Santarossa G, "Mutations in the “lid” region affect chain length specificity and thermostability of a Pseudomonas fragi lipase", FEBS Lett, 579, pp.2383~2386 (2005)
- Schuttelkopf AW, "PRODRG: a tool for high-throughput crystallography of protein-ligand complexes", Acta Crystallogr. D, 60, pp.1355~1363 (2004)
- Shih TW, "Substitution of Asp189 residue alters the activity and thermostability of Geobacillus sp. NTU 03 lipase", Biotechnol. Lett, 33, pp.1841~1846 (2011)
- Tang L, "Substitution of Val72 residue alters the enantioselectivity and activity of Penicillium expansum lipase", World J. Microbiol. Biotechnol, 29, pp.145~151 (2013)
- Verger R., "'Interfacial activation' of lipases: facts and artifacts", Trends Biotechnol, 15, pp.32~38 (1997)
- Wang WF, "Production of lipase SMG1 and its application in synthesizing diacylglyecrol", J. Mol. Catal. B Enzym, 77, pp.87~91 (2012)
- Wang XP, "Hydrolysis of lysophosphatidylcholines by a lipase from Malassezia globosa", Eur. J. Lipid Sci. Technol, pp.201400643~ (2015)[DOI]
- Xu D, "Enzymatic synthesis of diacylglycerols enriched with conjugated linoleic acid by a novel lipase from Malassezia globosa", J. Am. Oil Chem. Soc, 89, pp.1259~1266 (2012)
- Xu TT, "Crystal structure of a mono- and diacylglycerol lipase from Malassezia globosa reveals a novel lid conformation and insights into the substrate specificity", J. Struct. Biol, 178, pp.363~369 (2012)
- Yuan D, "Biochemical properties of a new cold-active mono- and diacylglycerol lipase from marine member Janibacter sp. strain HTCC2649", Int. J. Mol. Sci, 15, pp.10554~10566 (2014)