Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 276, Issue 40, Pages 37547-37555Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M105982200
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Funding
- NHLBI NIH HHS [R01 HL56773, F32 HL10170] Funding Source: Medline
- NIGMS NIH HHS [P01 GM57323] Funding Source: Medline
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Prostaglandin endoperoxide H synthases-1 and -2 (PGHSs) can oxygenate 18-22 carbon polyunsaturated fatty acids, albeit with varying efficiencies. Here we report the crystal structures of eicosapentaenoic acid (EPA, 20:5 n-3) and linoleic acid (LA, 18:2 n-6) bound in the cyclooxygenase active site of Co3+ protoporphyrin IX-reconstituted ovine PGHS-1 (Co3+-oPGHS-1) and compare the effects of active site substitutions on the rates of oxygenation of EPA, LA, and arachidonic acid (AA). Both EPA and IA bind in the active site with orientations similar to those seen previously with AA and dihomo-gamma -linolenic acid (DHLA). For EPA, the presence of an additional double bond (C-17/C-18) causes this substrate to bind in a strained conformation in which C-13 is misaligned with respect to Tyr-385, the residue that abstracts hydrogen from substrate fatty acids. Presumably, this misalignment is responsible for the low rate of EPA oxygenation. For LA, the carboxyl half binds in a more extended configuration than AA, which results in positioning C-11 next to Tyr-385. Val-349 and Ser-530, recently identified as important determinants for efficient oxygenation of DHLA by PGHS-1, play similar roles in the oxygenation of EPA and I-A. Approximately 750- and 175-fold reductions in the oxygenation efficiency of EPA and LA were observed with V349A oPGHS-1, compared with a 2-fold change for AA. Val-349 contacts C-2 and C-3 of EPA and C-4 of LA orienting the carboxyl halves of these substrates so that the omega -ends are aligned properly for hydrogen abstraction. An S530T substitution decreases the V-max/K-m of EPA and LA by 375- and 140-fold. Ser-530 makes six contacts with EPA and four with LA involving C-8 through C-16; these interactions influence the alignment of the substrate for hydrogen abstraction. Interestingly, replacement of Phe-205 increases the volume of the cyclooxygenase site allowing EPA to be oxygenated more efficiently than with native oPGHS-1.
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