title: Elucidation of a key position for acyltransfer activity in Candida parapsilosis lipase/acyltransferase (CpLIP2) and in Pseudozyma antarctica lipase A (CAL-A) by rational design.
abstract: Performing transesterifications in aqueous media is becoming a priority challenge in lipid biotechnology in order to develop more eco-friendly and efficient biocatalytic processes in systems containing both polar and apolar substrates. In this context, our group has explored for several years the high potential of the lipase/acyltransferase CpLIP2 from Candida parapsilosis and of several of its homologs, that catalyze efficiently acyltransfer reactions in lipid/water media with high water activity (aw>0.9). The discovery of a new member of this group, CduLAc from Candida dubliniensis, with a higher acyltransferase activity than CpLIP2, has provided a new insight on structure-function relationships in this group. Indeed, the comparison of sequences and 3D models, especially of CpLIP2 and CduLAc, with those of the phylogenetically related lipase A from Pseudozyma antarctica (CAL-A), allowed elucidating a key structural determinant of the acyltransferase activity: serine S369 in CpLIP2 and its equivalents E370 in CAL-A and A366 in CduLAc. Mutants obtained by rational design at this key position showed significant changes in acyltransfer activity. Whereas mutation S369E resulted in an increase in the hydrolytic activity of CpLIP2, S369A increased alcoholysis. More strikingly, the single E370A mutation in CAL-A drastically increased the acyltransferase activity of this enzyme, giving it the character of a lipase/acyltransferase. Indeed, this single mutation lowered the methanol concentration for which the initial rates of alcoholysis and hydrolysis are equal from 2M in CAL-A down to 0.3M in its mutant, while the exceptional stability of the parental enzyme toward alcohol and temperature was conserved.
authors.affiliation: Montpellier SupAgro, UMR 1208 IATE, 2 Place Viala, 34060 Montpellier cedex 2, France.
authors.forename: Anne-Hélène
authors.initials: AH
authors.lastname: Jan
authors.affiliation: Montpellier SupAgro, UMR 1208 IATE, 2 Place Viala, 34060 Montpellier cedex 2, 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, 34060 Montpellier cedex 2, France.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Deyrieux
authors.affiliation: Montpellier SupAgro, UMR 1208 IATE, 2 Place Viala, 34060 Montpellier cedex 2, France.
authors.forename: Véronique
authors.initials: V
authors.lastname: Perrier
authors.affiliation: Montpellier SupAgro, UMR 1208 IATE, 2 Place Viala, 34060 Montpellier cedex 2, France.
authors.forename: Éric
authors.initials: É
authors.lastname: Dubreucq
mesh_terms: D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics*; D000438:Alcohols / Q000737:chemistry; D001709:Biotechnology; D002175:Candida / Q000201:enzymology; D002384:Catalysis; D004951:Esterification / Q000235:genetics*; D008055:Lipids / Q000737:chemistry / Q000235:genetics; D020932:Nerve Growth Factor / Q000737:chemistry* / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D010802:Phylogeny; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014587:Ustilaginales / Q000201:enzymology; D014867:Water / Q000737:chemistry
chemical_list: D000438:Alcohols; C104081:Lip2 protein, rat; D008055:Lipids; D010446:Peptide Fragments; D014867:Water; D020932:Nerve Growth Factor; D000217:Acyltransferases
keywords: Biocatalysis; CAL-A; CpLIP2; Lipases/acyltransferases; Rational design; Transesterification