Identification and biochemical characterization of a GDSL-motif carboxylester hydrolase from Carica papaya latex.
An esterase (CpEst) showing high specific activities on tributyrin and short chain vinyl esters was obtained from Carica papaya latex after an extraction step with zwitterionic detergent and sonication, followed by gel filtration chromatography. Although the protein could not be purified to complete homogeneity due to its presence in high molecular mass aggregates, a major protein band with an apparent molecular mass of 41 kDa was obtained by SDS-PAGE. This material was digested with trypsin and the amino acid sequences of the tryptic peptides were determined by LC/ESI/MS/MS. These sequences were used to identify a partial cDNA (679 bp) from expressed sequence tags (ESTs) of C. papaya. Based upon EST sequences, a full-length gene was identified in the genome of C. papaya, with an open reading frame of 1029 bp encoding a protein of 343 amino acid residues, with a theoretical molecular mass of 38 kDa. From sequence analysis, CpEst was identified as a GDSL-motif carboxylester hydrolase belonging to the SGNH protein family and four potential N-glycosylation sites were identified. The putative catalytic triad was localised (Ser(35)-Asp(307)-His(310)) with the nucleophile serine being part of the GDSL-motif. A 3D-model of CpEst was built from known X-ray structures and sequence alignments and the catalytic triad was found to be exposed at the surface of the molecule, thus confirming the results of CpEst inhibition by tetrahydrolipstatin suggesting a direct accessibility of the inhibitor to the active site.
Abdelkafi
Slim
S
CNRS, Aix-Marseille Université, Enzymologie Interfaciale et Physiologie de la Lipolyse, Marseille, France.
Ogata
Hiroyuki
H
Barouh
Nathalie
N
Fouquet
Benjamin
B
Lebrun
Régine
R
Pina
Michel
M
Scheirlinckx
Frantz
F
Villeneuve
Pierre
P
Carrière
Frédéric
F
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D029441:Carica / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007783:Lactones / Q000494:pharmacology; D007840:Latex / Q000737:chemistry*; D008049:Lipase / Q000037:antagonists & inhibitors; D008066:Lipolysis / Q000187:drug effects; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D000077403:Orlistat; D010802:Phylogeny; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D012995:Solubility / Q000187:drug effects; D013379:Substrate Specificity / Q000187:drug effects
D007783:Lactones; D007840:Latex; D000077403:Orlistat; D004950:Esterases; D008049:Lipase