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Research article

Article

Research article

J. Microbiol. Biotechnol. 2015; 25(11): 1827-1834

Published online November 28, 2015 https://doi.org/10.4014/jmb.1506.06079

Copyright © The Korean Society for Microbiology and Biotechnology.

The Role of Residues 103, 104, and 278 in the Activity of SMG1 Lipase from Malassezia globosa: A Site-Directed Mutagenesis Study

Dongming Lan 1, Qian Wang 2, Grzegorz Maria Popowicz 3, Bo Yang 2, Qingyun Tang 1 and Yonghua Wang 1*

1College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510641, P.R. China, 2School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, P.R. China, 3Institute of Structural Biology, Helmholtz Zentrum München, Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH), D-85764 Neuherberg, Germany

Received: July 1, 2015; Accepted: August 4, 2015

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.

Keywords: lipase, substrate selectivity, lid, site-directed mutation