title: The 3D model of the lipase/acyltransferase from Candida parapsilosis, a tool for the elucidation of structural determinants in CAL-A lipase superfamily.
abstract: Because lipids are hydrophobic, the development of efficient bioconversions in aqueous media free of organic solvents is particularly challenging for green oleochemistry. Within this aim, enzymes exhibiting various abilities to catalyze acyltransfer reaction in water/lipid systems have been identified. Among these, CpLIP2 from Candida parapsilosis has been characterized as a lipase/acyltransferase, able to catalyze acyltransfer reactions preferentially to hydrolysis in the presence of particularly low acyl acceptor concentration and high thermodynamic activity of water (aw>0.9). Lipase/acyltransferases are thus of great interest, being able to produce new esters at concentrations above the thermodynamic equilibrium of hydrolysis/esterification with limited to no release of free fatty acids. Here, we present a 3D model of CpLIP2 based on homologies with crystallographic structures of Pseudozyma antarctica lipase A. Indeed, the two enzymes have 31% of identity in their primary sequence, yielding a same general structure, but different catalytic properties. The quality of the calculated CpLIP2 model was confirmed by several methods. Limited proteolysis confirmed the location of some loops at the surface of the protein 3D model. Directed mutagenesis also supported the structural model constructed on CAL-A template: the functional properties of various mutants were consistent with their structure-based putative involvement in the oxyanion hole, substrate specificity, acyltransfer or hydrolysis catalysis and structural stability. The CpLIP2 3D model, in comparison with CAL-A 3D structure, brings insights for the elucidation and improvement of the structural determinants involved in the exceptional acyltransferase properties of this promising biocatalyst and of homologous enzymes of the same family.
authors.affiliation: Montpellier SupAgro, UMR 1208 IATE, 2 Place Viala, F-34060 Montpellier cedex, France. Electronic address: maeva.subileau@supagro.fr.
authors.forename: Maeva
authors.initials: M
authors.lastname: Subileau
authors.affiliation: Montpellier SupAgro, UMR 1208 IATE, 2 Place Viala, F-34060 Montpellier cedex, France.
authors.forename: Anne-Hélène
authors.initials: AH
authors.lastname: Jan
authors.affiliation: iBiTec-S, Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), Commissariat à l'énergie atomique et aux énergies alternatives (CEA) Saclay, France.
authors.forename: Hervé
authors.initials: H
authors.lastname: Nozac'h
authors.affiliation: IIS-Fundación Jiménez Díaz, Department of Allergy Avda. Reyes Católicos, 2, Madrid, ES 28040, Spain.
authors.forename: Marina
authors.initials: M
authors.lastname: Pérez-Gordo
authors.affiliation: Montpellier SupAgro, UMR 1208 IATE, 2 Place Viala, F-34060 Montpellier cedex, France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Perrier
authors.affiliation: Montpellier SupAgro, UMR 1208 IATE, 2 Place Viala, F-34060 Montpellier cedex, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Dubreucq
mesh_terms: D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D002175:Candida / Q000737:chemistry* / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006868:Hydrolysis; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D010169:Palmitic Acids / Q000737:chemistry*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D010169:Palmitic Acids; D011994:Recombinant Proteins; D000217:Acyltransferases; D008049:Lipase; C007680:ethyl palmitate
keywords: Biocatalysis; CAL-A; CpLIP2; Green chemistry; Lipase/acyltransferase; Protein engineering