tailieunhanh - Báo cáo khóa học: Structural and functional comparison of 15S- and 15R-specific cyclooxygenases from the coral Plexaura homomalla

It has been known for 30 years that the gorgonian coral Plexaura homomallacontains either 15S-or 15R-configur-ation prostaglandins (PGs), depending on its location in the Caribbean. Recently we showed that the 15R-PGs in the R-variety of P. homomallaare formed by a unique cyclo-oxygenase (COX) with 15Roxygenation specificity [Valm-sen, K., Ja¨rving, I., Boeglin, ., Varvas, K., Koljak, R., Pehk, T., Brash, . & Samel, N. (2001)Proc. Natl. Acad. Sci. USA98, 7700]. Here we describe the cloning and char-acterization of a closely related COX protein (97% amino acid sequence identity) from theS-variety ofP. homomalla | Eur. J. Biochem. 271 3533-3538 2004 FEBS 2004 doi Structural and functional comparison of 155- and 15 -specific cyclooxygenases from the coral Plexaura homomalla Karin Valmsen1 William E. Boeglin2 Ivar Jarving1 Claus Schneider2 Kulliki Varvas1 Alan R. Brash2 and Nigulas Samel1 1 Department of Chemistry Tallinn University of Technology Estonia department of Pharmacology Vanderbilt University School of Medicine Nashville TN USA It has been known for 30 years that the gorgonian coral Plexaura homomalla contains either 15S- or 15R-configur-ation prostaglandins PGs depending on its location in the Caribbean. Recently we showed that the 15R-PGs in the R-variety of P. homomalla are formed by a unique cyclooxygenase COX with 15R oxygenation specificity Valm-sen K. Jarving I. Boeglin . Varvas K. Koljak R. Pehk T. Brash . Samel N. 2001 Proc. Natl. Acad. Sci. USA 98 7700 . Here we describe the cloning and characterization of a closely related COX protein 97 amino acid sequence identity from the S-variety of P. homomalla. Functional expression of the S-variant COX cDNA in Sf9 insect cells followed by incubation with exogenous arachidonic acid resulted in formation of PG products with 98 15S-configuration. Mutational analysis was performed on a suggested active site determinant of C-15 oxygenation specificity position 349 Val in all S-specific cOX Ile in 15R-COX . The 15S-COX Val349 to Ile mutant formed 35 15R-PGs while the reverse mutation in the 15R-COX Ile349Val led to formation of 70 15S-pro ducts. This establishes position 349 as an important determinant of the product stereochemistry at C-15. Our characterization of the enzyme variants demonstrates that very minor sequence divergence accounts for the content of epimeric PGs in the two variants of P. homomalla and that the differences do not arise by isomerization of the products. Keywords cyclooxygenase Plexaura homomalla 15R-pro-staglandins site directed mutagenesis .