title: Biochemical characterization of Yarrowia lipolytica LIP8, a secreted lipase with a cleavable C-terminal region.
abstract: Yarrowia lipolytica is a lipolytic yeast possessing 16 paralog genes coding for lipases. Little information on these lipases has been obtained and only the major secreted lipase, namely YLLIP2, had been biochemically and structurally characterized. Another secreted lipase, YLLIP8, was isolated from Y. lipolytica culture medium and compared with the recombinant enzyme produced in Pichia pastoris. N-terminal sequencing showed that YLLIP8 is produced in its active form after the cleavage of a signal peptide. Mass spectrometry analysis revealed that YLLIP8 recovered from culture medium lacks a C-terminal part of 33 amino acids which are present in the coding sequence. A 3D model of YLLIP8 built from the X-ray structure of the homologous YLLIP2 lipase shows that these truncated amino acids in YLLIP8 belong to an additional C-terminal region predicted to be mainly helical. Western blot analysis shows that YLLIP8 C-tail is rapidly cleaved upon enzyme secretion since both cell-bound and culture supernatant lipases lack this extension. Mature recombinant YLLIP8 displays a true lipase activity on short-, medium- and long-chain triacylglycerols (TAG), with an optimum activity at alkaline pH on medium chain TAG. It has no apparent regioselectivity in TAG hydrolysis, thus generating glycerol and FFAs as final lipolysis products. YLLIP8 properties are distinct from those of the 1,3-regioselective YLLIP2, acting optimally at acidic pH. These lipases are tailored for complementary roles in fatty acid uptake by Y. lipolytica.
authors.affiliation: University of Sfax, ENIS - Laboratory of Biochemistry and Enzymatic Engineering of Lipases, 3038 Sfax, Tunisia.
authors.forename: Jannet
authors.initials: J
authors.lastname: Kamoun
authors.affiliation: Proteabio Europe S.A.S., 290 Chemin de Saint-Dionysy, Bât A, 30980 Langlade, France.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Schué
authors.affiliation: University of Sfax, ENIS - Laboratory of Biochemistry and Enzymatic Engineering of Lipases, 3038 Sfax, Tunisia.
authors.forename: Wala
authors.initials: W
authors.lastname: Messaoud
authors.affiliation: Proteabio Europe S.A.S., 290 Chemin de Saint-Dionysy, Bât A, 30980 Langlade, France.
authors.forename: Justine
authors.initials: J
authors.lastname: Baignol
authors.affiliation: CNRS, Aix-Marseille Université, UMR 7282 Enzymologie Interfaciale et Physiologie de la Lipolyse, 31 Chemin Joseph Aiguier, 13009 Marseille, France.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Point
authors.affiliation: CNRS, Aix-Marseille Université, UMR 7282 Enzymologie Interfaciale et Physiologie de la Lipolyse, 31 Chemin Joseph Aiguier, 13009 Marseille, France.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Mateos-Diaz
authors.affiliation: CNRS - Aix-Marseille Université, Plate-forme Protéomique FR3479, IBiSA Marseille-Protéomique, 31 Chemin Joseph Aiguier, 13009 Marseille, France.
authors.forename: Pascal
authors.initials: P
authors.lastname: Mansuelle
authors.affiliation: University of Sfax, ENIS - Laboratory of Biochemistry and Enzymatic Engineering of Lipases, 3038 Sfax, Tunisia.
authors.forename: Youssef
authors.initials: Y
authors.lastname: Gargouri
authors.affiliation: CNRS, Aix-Marseille Université, UMR 7282 Enzymologie Interfaciale et Physiologie de la Lipolyse, 31 Chemin Joseph Aiguier, 13009 Marseille, France.
authors.forename: Goetz
authors.initials: G
authors.lastname: Parsiegla
authors.affiliation: CNRS, Aix-Marseille Université, UMR 7282 Enzymologie Interfaciale et Physiologie de la Lipolyse, 31 Chemin Joseph Aiguier, 13009 Marseille, France.
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Cavalier
authors.affiliation: CNRS, Aix-Marseille Université, UMR 7282 Enzymologie Interfaciale et Physiologie de la Lipolyse, 31 Chemin Joseph Aiguier, 13009 Marseille, France. Electronic address: carriere@imm.cnrs.fr.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Carrière
authors.affiliation: University of Sfax, ENIS - Laboratory of Biochemistry and Enzymatic Engineering of Lipases, 3038 Sfax, Tunisia; CNRS, Aix-Marseille Université, UMR 7282 Enzymologie Interfaciale et Physiologie de la Lipolyse, 31 Chemin Joseph Aiguier, 13009 Marseille, France. Electronic address: ahmed.aloulou@enis.rnu.tn.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Aloulou
mesh_terms: D000595:Amino Acid Sequence; D001647:Bile Acids and Salts / Q000378:metabolism; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D005230:Fatty Acids, Nonesterified / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005990:Glycerol / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008066:Lipolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D000069463:Olive Oil; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D010938:Plant Oils / Q000378:metabolism; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014280:Triglycerides / Q000378:metabolism; D025062:Yarrowia / Q000201:enzymology* / Q000235:genetics
chemical_list: D001647:Bile Acids and Salts; D005230:Fatty Acids, Nonesterified; D005656:Fungal Proteins; D000069463:Olive Oil; D010938:Plant Oils; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D014280:Triglycerides; D008049:Lipase; D005990:Glycerol
keywords: Enzyme; Lipolysis; Pichia pastoris; Regioselectivity; Signal peptide; Yeast