Homology modeling and biochemical characterization of substrate binding to cyclooxygenase - peroxidase superfamily members Aspergillus fumigatus 5,8-linoleate diol synthase and Gracilaria vermiculophylla cyclooxygenase
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This thesis is focused on representative members of the Cyclooxygenase-Peroxidase (COX-POX) superfamily of enzymes. The members of this superfamily present structural conservation of heme binding motifs, as well as elements involved in the oxygenation of fatty acid (FA) substrates to form physiologically important oxylipin products. Well-characterized members include the animal cyclooxygenase (COX) and the plant α-dioxygenase (α-DOX) enzymes. Despite their structural similarities, the mammalian COX isozymes (COX-1 and COX-2) differ in the role of Arg-120 in high affinity substrate binding and positioning. An ionic interaction between the guanidinium group of Arg-120 and the carboxylate moiety of the substrate arachidonic acid (AA) is required for high affinity binding and catalysis in COX-1. This interaction is not required for COX-2 catalysis. We probed the role of residues lining the hydrophobic pocket of the COX-2 cyclooxygenase active site in high affinity substrate binding and positioning. Results indicate that a concerted effort from the hydrophobic residues lining the active site are likely required for high affinity AA binding. We also utilized homology modeling and biochemical assays to study substrate binding and catalysis in the COX-POX members Aspergillus fumigatus ( A.f ) 5,8-linoleate diol synthase (5,8-LDS) and Gracilaria vermiculophylla ( G.v ) COX. Through homology modeling, we identified residues that may play roles in substrate binding and oxygenation. Our results indicate that high affinity substrate binding in 5,8-LDS and G.v COX may be governed by hydrophobic residues within the active site, similar to COX-2. Ser-530, Leu-531, and Gly-533 play important roles in substrate positioning and product stereochemistry in COX. We probed the roles of equivalent residues in G.v COX. Substitutions at these positions in G.v COX greatly affected AA oxygenation, implicating the importance of these residues in catalysis despite evolutionary variation between the COX.