| Summary: | 碩士 === 臺灣大學 === 生物化學暨分子生物學研究所 === 98 === Thromboxane synthase (TXAS) and prostacyclin synthase(PGIS) belong to the heme-containing cytochrome P450 superfamily that participates in numerous crucial oxidation processes. However, rather than possessing a monooxygenation activity like most other P450 enzymes, PGIS and TXAS carry out isomerization reactions and require neither NAPH nor O2 for function, they are classified as class III P450. Interestingly, while prostaglandin H2 (PGH2) is a common substrate for both enzymes, thromboxane A2 (TXA2) and prostacyclin (PGI2) are produced by TXAS and PGIS, respectively. Spectroscopic studies indicated that stereospecific substrate binding determines product specificity. In TXAS, the O-9 of PGH2 ligates to the heme-iron, but in PGIS the O-11 of PGH2 serves as the heme ligand. Although TXAS and PGIS catalyze chemical conversion of the same substrate (PGH2), their respective product displays opposite biological activities. TXA2 is a potent stimulator of vasoconstriction and platelet activation. In contrast, PGI2 inhibits vasoconstriction and platelet aggregation. Together, these two bioactive prostanoids mediate key events in atherosclerosis, shock syndromes and carcinogenesis.
To understand how the stereospecific PGH2 binding is achieved, our lab has previously determined the crystal structures of PGIS and its complexes with substrate analog and inhibitor. Structural comparison has lead to the identification of residues in the catalytic center that may be crucial for catalysis. To understand these residues catalytic roles in greater detail, residues W272 and N277 have been mutated as alanines, and crystallographic analyses are performed to examine how substrate binding is affected by mutations. In addition, we attempted to initiate structural studies on TXAS to gain more insights on the molecular basis of stereospecific substrate recognition.
The crystal structures of ligand-free and substrate analog-bound PGIS-N277A mutant were determined at 2.37 Å and 2.57 Å, respectively. The structure around the enzyme active site reveals that the hydrogen bond between N277 side chain and O-9 of PGH2 was abolished upon mutating N277 to Alanine. However, the spatial orientation of the substrate analog and the arrangement of surrounding residues are not altered. Unexpectedly, the W272A mutation alters the conformation of N277 side chain, leading to the loss of hydrogen bond between N277 and O-9 of PGH2. These observations support the potential importance of W272 and N277 in catalysis and are consistent with the highly conserved nature of these residues. To obtain soluble protein samples for crystallization, TXAS was modified by replacing the N-terminal transmembrane domain with a hydrophilic sequence, MAKKTSS. Nevertheless, homogeneity and yield of purified mouse TXAS remain to be improved before crystallization trials can be performed.
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