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PDBsum entry 1igz

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Oxidoreductase PDB id
1igz
Jmol
Contents
Protein chain
553 a.a. *
Ligands
NAG-NDG
NAG-NAG-BMA-BMA-
MAN
NAG-NAG
BGC
BOG ×2
COH
EIC
Waters ×84
* Residue conservation analysis

References listed in PDB file
Key reference
Title Structure of eicosapentaenoic and linoleic acids in the cyclooxygenase site of prostaglandin endoperoxide h synthase-1.
Authors M.G.Malkowski, E.D.Thuresson, K.M.Lakkides, C.J.Rieke, R.Micielli, W.L.Smith, R.M.Garavito.
Ref. J Biol Chem, 2001, 276, 37547-37555. [DOI no: 10.1074/jbc.M105982200]
PubMed id 11477109
Abstract
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 Co(3+) protoporphyrin IX-reconstituted ovine PGHS-1 (Co(3+)-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 LA 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 LA. 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.
Figure 1.
Fig. 1. EPA bound in the cyclooxygenase active site of the Co3+-oPGHS-1·EPA complex. A, stereo view of EPA bound in the COX channel. The initial positive F[o] F[c] electron density (purple) within the COX channel, contoured at 2.0 , is shown with the final refined model of EPA (light blue). Side chain atoms for residues that predominantly contact the substrate at the carboxylate, C-2 through C-14, and C-15 through C-20 are colored blue, red, and green, respectively. Ser-530 (purple) lies below Tyr-385 (orange). The side chain of Ser-353 has been omitted for clarity. Single-letter amino acid codes are used throughout figures. B, a view of the cyclooxygenase active site rotated 90° about the vertical axis using the same color scheme as in A. The positive F[o] F[c] "omit" electron density (red) contoured at 2.0 for EPA is shown. All figures were created fully or in part using the program SETOR (68).
Figure 4.
Fig. 4. Comparison of the binding of EPA and AA within the cyclooxygenase active site. A, stereo view of EPA (light blue) and AA (yellow) overlapped in the cyclooxygenase active site. Active site residues are colored as in Fig. 1, and modeled hydrogen atoms on C-13 of both substrates are shown. B, stereo view of the -end of EPA and AA from the perspective of Tyr-385 at the top of the cyclooxygenase active site (rotation of 90° about the horizontal axis in A).
The above figures are reprinted by permission from the ASBMB: J Biol Chem (2001, 276, 37547-37555) copyright 2001.
Secondary reference #1
Title Mutational and X-Ray crystallographic analysis of the interaction of dihomo-Gamma -Linolenic acid with prostaglandin endoperoxide h synthases.
Authors E.D.Thuresson, M.G.Malkowski, K.M.Lakkides, C.J.Rieke, A.M.Mulichak, S.L.Ginell, R.M.Garavito, W.L.Smith.
Ref. J Biol Chem, 2001, 276, 10358-10365. [DOI no: 10.1074/jbc.M009378200]
PubMed id 11121413
Full text Abstract
Figure 2.
Fig. 2. Comparison of the binding of AA and DHLA within the cyclooxygenase active site. A stereo view of DHLA (red) and AA (light blue) (7) bound within in the cyclooxygenase active site channel of oPGHS-1. Active site residues are colored as in Fig. 1. The absence of the C5/C6 double bond in DHLA allows for greater conformational flexibility in the carboxyl half of the substrates as compared with AA. This is reflected in the 1.1-Å r.m.s. deviation between carbon positions in DHLA versus AA for C-1 to C-10. Additionally, the position of the C -2 atom of Ile-523 (orange in DHLA versus blue in AA) and the O atom on Ser-530 (light green in DHLA versus magenta in AA) move to accommodate DHLA in the active site.
Figure 3.
Fig. 3. Interactions between DHLA and cyclooxygenase active site residues. A schematic diagram of the interactions between DHLA and residues within the cyclooxygenase channel. Every other carbon atom of DHLA is labeled, and the hydrogens for C-13 have been modeled. All dashed lines represent interactions within 4.0 Å between the specific side chain atom of the protein and DHLA. Only 3 of the 62 contacts between DHLA and cyclooxygenase channel residues are hydrophilic.
The above figures are reproduced from the cited reference with permission from the ASBMB
Secondary reference #2
Title The productive conformation of arachidonic acid bound to prostaglandin synthase.
Authors M.G.Malkowski, S.L.Ginell, W.L.Smith, R.M.Garavito.
Ref. Science, 2000, 289, 1933-1937. [DOI no: 10.1126/science.289.5486.1933]
PubMed id 10988074
Full text Abstract
Figure 3.
Fig. 3. A schematic of interactions between AA and COX channel residues (30). Carbon atoms of AA are yellow, oxygen atoms red, and the 13proS hydrogen blue. All dashed lines represent interactions within 4.0 Å between the specific side chain atom of the protein and AA; the structures AA-1 and AA-2 revealed the same set of 49 contacts. Only two of these contacts between AA and the COX channel residues are hydrophilic. The carboxylate forms a salt bridge to the guanidinium atom of Arg120 (distance = 2.4 Å; angle = 143°) and a hydrogen bond to the OH group of Tyr355 (distance = 3.1 Å; angle = 115°). (Inset) A schematic of the chemical structure of AA.
Figure 4.
Fig. 4. Mechanistic sequence for converting AA to PGG[2] (30). Abstraction of the 13-proS hydrogen by the tyrosyl radical leads to the migration of the radical to C-11 on AA. Attack of molecular oxygen, coming from the base of the COX channel, occurs on the side antarafacial to hydrogen abstraction. As the 11R-peroxyl radical swings over C-8 for an R-side attack on C-9 to form the endoperoxide bridge, C-12 is brought closer to C-8 via rotation about the C-10/C-11 bond allowing the formation of the cyclopentane ring. The movement of C-12 also positions C-15 optimally for addition of a second molecule of oxygen, formation of PGG[2], and the migration of the radical back to Tyr385.
The above figures are reproduced from the cited reference with permission from the AAAs
PROCHECK
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