title: The Role of Residues 103, 104, and 278 in the Activity of SMG1 Lipase from Malassezia globosa: A Site-Directed Mutagenesis Study.
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.
authors.affiliation: College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510641, P.R. China.
authors.forename: Dongming
authors.initials: D
authors.lastname: Lan
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, P.R. China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Institute of Structural Biology, Helmholtz Zentrum München, Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH), D-85764 Neuherberg, Germany.
authors.forename: Grzegorz Maria
authors.initials: GM
authors.lastname: Popowicz
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, P.R. China.
authors.forename: Bo
authors.initials: B
authors.lastname: Yang
authors.affiliation: College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510641, P.R. China.
authors.forename: Qingyun
authors.initials: Q
authors.lastname: Tang
authors.affiliation: College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510641, P.R. China.
authors.forename: Yonghua
authors.initials: Y
authors.lastname: Wang
mesh_terms: D001665:Binding Sites; D004252:DNA Mutational Analysis; D004075:Diglycerides / Q000378:metabolism; D015870:Gene Expression; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008290:Malassezia / Q000201:enzymology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation
chemical_list: D004075:Diglycerides; D050505:Mutant Proteins; D008049:Lipase
keywords: lid; lipase; site-directed mutation; substrate selectivity