Study of a sterol esterase secreted by Ophiostoma piceae: sequence, model and biochemical properties.
An extracellular sterol esterase from Ophiostoma piceae efficiently hydrolyzes sterol esters, triglycerides and p-nitrophenol esters. cDNA was screened with a probe obtained by PCR using as primers oligonucleotides corresponding to the N-terminal and internal mature enzyme sequences and complete sequence was obtained by 3' rapid amplification of cDNA end (RACE) and inverse PCR. The O. piceae esterase gene had a length of 1.8 kbp and lacked introns. A search for proteins with related amino acid sequences revealed around 40% identity with lipases from Candida rugosa and Geotrichum candidum. Modelling the O. piceae enzyme, using the crystal structures of Lip1 and Lip3 from C. rugosa as templates, revealed a similar substrate-binding site, but some changes affecting the flap zone and the aromatic region of the tunnel may be responsible for the wide substrate specificity of this interesting sterol esterase. The ability of the new fungal esterase to hydrolyze triglycerides and esters of p-nitrophenol and cholesterol was compared with those of commercial lipases and cholesterol esterases showing the new enzyme the highest efficiency hydrolyzing triglycerides and sterol esters in the conditions assayed (in presence of Genapol X-100). Finally, the O. piceae gene was successfully expressed in Pichia pastoris, as a model organism to express fungal enzymes, resulting in higher levels of esterase activity than those obtained in the O. piceae cultures. In spite of its higher glycosylation degree, the recombinant enzyme was able to hydrolyze more efficiently than native enzyme the assayed substrates.
Calero-Rueda
Olga
O
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, E-28040 Madrid, Spain.
Barba
Víctor
V
Rodríguez
Enrique
E
Plou
Francisco
F
Martínez
Angel T
AT
Martínez
María Jesús
MJ
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D008958:Models, Molecular; D008969:Molecular Sequence Data; D055156:Ophiostoma / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D002787:Sterol Esterase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D002787:Sterol Esterase