The origin of 15 R -prostaglandins in the Caribbean coral Plexaura homomalla : Molecular cloning and expression of a novel cyclooxygenase

Abstract

The highest concentrations of prostaglandins in nature are found in the Caribbean gorgonian Plexaura homomalla. Depending on its geographical location, this coral contains prostaglandins with typical mammalian stereochemistry (15S-hydroxy) or the unusual 15R-prostaglandins. Their metabolic origin has remained the subject of mechanistic speculations for three decades. Here, we report the structure of a type of cyclooxygenase (COX) that catalyzes transformation of arachidonic acid into 15R-prostaglandins. Using a homology-based reverse transcriptase-PCR strategy, we cloned a cDNA corresponding to a COX protein from the R variety of P. homomalla. The deduced peptide sequence shows 80% identity with the 15S-specific coral COX from the Arctic soft coral Gersemia fruticosa and Ϸ50% identity to mammalian COX-1 and COX-2. The predicted tertiary structure shows high homology with mammalian COX isozymes having all of the characteristic structural units and the amino acid residues important in catalysis. Some structural differences are apparent around the peroxidase active site, in the membrane-binding domain, and in the pattern of glycosylation. When expressed in Sf9 cells, the P. homomalla enzyme forms a 15R-prostaglandin endoperoxide together with 11R-hydroxyeicosatetraenoic acid and 15R-hydroxyeicosatetraenoic acid as byproducts. The endoperoxide gives rise to 15R-prostaglandins and 12R-hydroxyheptadecatrienoic acid, identified by comparison to authentic standards. Evaluation of the structural differences of this 15R-COX isozyme should provide new insights into the substrate binding and stereospecificity of the dioxygenation reaction of arachidonic acid in the cyclooxygenase active site.

Developmental expression of functional cyclooxygenases in zebrafish

“…2). The zebrafish sequences locate to the respective arms of the other vertebrate COX-1 and COX-2 enzymes, which are separated from the invertebrate COX of the coral species Gersemia fruticosa (20) and Plexaura homomalla (21). Parsimony analysis confirmed the branch topology of the distance phylogeny, indicating that mammalian and teleost COXs were indeed sister groups (data not shown).…”

Biosynthesis and function of polyacetylenes and allied natural products

“…The unusual 1,2-dioxane rings of these chiral structures may be formed by a cyclooxygenase-like reaction, where closure of the peroxide occurs at a ketone. Examples of COX-1 and COX-2 have been cloned and heterologously expressed from the corals Gersemia fruticosa and Plexaura homomalla [364,365]. Amazingly, a pair of R-and S-stereospecific P. homomalla COX-2 isozymes showed 97% identity at the amino acid level and had seven highly conserved residues when compared to known cyclooxygenases; a single substitution of Ile-349 for Val in the substrate binding channel switched the stereochemistry from 15R to 15S for 70% of the prostaglandin produced [366].…”

8R-Lipoxygenase-catalyzed synthesis of a prominent cis-epoxyalcohol from dihomo-γ-linolenic acid: a distinctive transformation compared with S-lipoxygenases

“…There is little doubt that this product and our epoxyalcohol are the same compound. The existence of 8 R -LOX metabolism in P. homomalla was not uncovered until the mid-1980s, a decade after these early biosynthetic studies ( 8 ), and it was only around the years 1995-2000 that the origin of the coral prostaglandins via cyclooxygenase was fi rmly established ( 6,7,(47)(48)(49). (8,9-cis -epoxy, 9,10 erythro ) and the complete retention of the hydroperoxy oxygens.…”