期刊
EUROPEAN JOURNAL OF BIOCHEMISTRY
卷 271, 期 17, 页码 3533-3538出版社
WILEY
DOI: 10.1111/j.0014-2956.2004.04289.x
关键词
cyclooxygenase; Plexaura homomalla; 15R-prostaglandins; site directed mutagenesis; stereospecificity
资金
- NIGMS NIH HHS [GM-53638] Funding Source: Medline
It has been known for 30 years that the gorgonian coral Plexaura homomalla contains either 15S- or 15R-configuration 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 [Valmsen, K., Jarving, I., Boeglin, W.E., Varvas, K., Koljak, R., Pehk, T., Brash, A.R. & Samel, N. (2001) Proc. Natl. Acad. Sci. USA98, 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-products. 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.
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