A critical role of non-active site residues on cyclooxygenase helices 5 and 6 in the control of prostaglandin stereochemistry at carbon 15

The correct stereochemistry of prostaglandins is a prerequisite of their biological activity and thus is under a strict enzymatic control. Recently, we cloned and characterized two cyclooxygenase (COX) isoforms in the coral Plexaura homomalla that share 97% amino acid sequence identity, yet form prostaglandins with opposite stereochemistry at carbon 15. The difference in oxygenation specificity is only partially accounted for by the single amino acid substitution in the active site (Ile or Val at position 349). For further elucidation of residues involved in the C-15 stereocontrol, a series of sequence swapping and site-directed mutagenesis experiments between 15R- and 15S-COX were performed. Our results show that the change in stereochemistry at carbon 15 of prostaglandins relates mainly to five amino acid substitutions on helices 5 and 6 of the coral COX. In COX proteins, these helices form a helix-turn-helix motif that traverses through the entire protein, contributing to the second shell of residues around the oxygenase active site; it constitutes the most highly conserved region where even slight changes result in loss of catalytic activity. The finding that this region is among the least conserved between the P. homomalla 15S- and 15R-specific COX further supports its significance in maintaining the desired prostaglandin stereochemistry at C-15. The results are particularly remarkable because, based on its strong conservation, the conserved middle of helix 5 is considered as central to the core structure of peroxidases, of which COX proteins are derivatives. Now we show that the same parts of the protein are involved in the control of oxygenation with 15R or 15S stereospecificity in the dioxygenase active site.     

Identification of two cyclooxygenase active site residues, Leucine 384 and Glycine 526, that control carbon ring cyclization in prostaglandin biosynthesis

The cyclooxygenase (COX) reaction of prostaglandin (PG) biosynthesis begins with the highly specific oxygenation of arachidonic acid in the 11R configuration and ends with a 15S oxygenation to form PGG2. To obtain new insights into the mechanisms of stereocontrol of oxygenation, we mutated active site residues of human COX-2 that have potential contacts with C-11 of the reacting substrate. Although the 11R oxygenation was not perturbed, changing Leu-384 (into Phe, Trp), Trp-387 (Phe, Tyr), Phe-518 (Ile, Trp, Tyr), and Gly-526 (Ala, Ser, Thr, Val) impaired or abrogated PGG2 synthesis, and typically 11R-HETE was the main product formed. The Gly-526 and Leu-384 mutants formed, in addition, three novel products identified by LC-MS, NMR, and circular dichroism as 8,9-11,12-diepoxy-13R-(or 15R)-hydro(pero)xy derivatives of arachidonic acid. Mechanistically, we propose these arise from a free radical intermediate in which a C-8 carbon radical displaces the 9,11-endoperoxide O-O bond to yield an 8,9-11,12-diepoxide that is finally oxygenated stereospecifically in the 13R or 15R configuration. Formation of these novel products signals an arrest in the normal course of prostaglandin synthesis just prior to closing of the 5-membered carbon ring, and points to a crucial role for Leu-384 and Gly-526 in the correct positioning of the reacting fatty acid intermediate. Some of the Gly-526 and Leu-384 mutants catalyzed both formation of PGG2 (with the normal 15S configuration) and the 13R- or 15R-oxygenated diepoxides. This result suggests that oxygenation specificity can be determined by the orientation of the reacting fatty acid radical and is not a predetermined outcome based solely on the structure of the cyclooxygenase active site.     

Catalytic convergence of manganese and iron lipoxygenases by replacement of a single amino Acid

Lipoxygenases (LOXs) contain a hydrophobic substrate channel with the conserved Gly/Ala determinant of regio- and stereospecificity and a conserved Leu residue near the catalytic non-heme iron. Our goal was to study the importance of this region (Gly(332), Leu(336), and Phe(337)) of a lipoxygenase with catalytic manganese (13R-MnLOX). Recombinant 13R-MnLOX oxidizes 18:2n-6 and 18:3n-3 to 13R-, 11(S or R)-, and 9S-hydroperoxy metabolites (～80-85, 15-20, and 2-3%, respectively) by suprafacial hydrogen abstraction and oxygenation. Replacement of Phe(337) with Ile changed the stereochemistry of the 13-hydroperoxy metabolites of 18:2n-6 and 18:3n-3 (from ～100% R to 69-74% S) with little effect on regiospecificity. The abstraction of the pro-S hydrogen of 18:2n-6 was retained, suggesting antarafacial hydrogen abstraction and oxygenation. Replacement of Leu(336) with smaller hydrophobic residues (Val, Ala, and Gly) shifted the oxygenation from C-13 toward C-9 with formation of 9S- and 9R-hydroperoxy metabolites of 18:2n-6 and 18:3n-3. Replacement of Gly(332) and Leu(336) with larger hydrophobic residues (G332A and L336F) selectively augmented dehydration of 13R-hydroperoxyoctadeca-9Z,11E,15Z-trienoic acid and increased the oxidation at C-13 of 18:1n-6. We conclude that hydrophobic replacements of Leu(336) can modify the hydroperoxide configurations at C-9 with little effect on the R configuration at C-13 of the 18:2n-6 and 18:3n-3 metabolites. Replacement of Phe(337) with Ile changed the stereospecific oxidation of 18:2n-6 and 18:3n-3 with formation of 13S-hydroperoxides by hydrogen abstraction and oxygenation in analogy with soybean LOX-1.     

Identification of amino acid determinants of the positional specificity of mouse 8S-lipoxygenase and human 15S-lipoxygenase-2

Phorbol ester-inducible mouse 8S-lipoxygenase (8-LOX) and its human homologue, 15S-lipoxygenase-2 (15-LOX-2), share 78% identity in amino acid sequences, yet there is no overlap in their positional specificities. In this study, we investigated the determinants of positional specificity using a random chimeragenesis approach in combination with site-directed mutagenesis. Exchange of the C-terminal one-third of the 8-LOX with the corresponding portion of 15-LOX-2 yielded a chimeric enzyme with exclusively 15S-lipoxygenase activity. The critical region was narrowed down to a cluster of five amino acids by expression of multiple cDNAs obtained by in situ chimeragenesis in Escherichia coli. Finally, a pair of amino acids, Tyr(603) and His(604), was identified as the positional determinant by site-directed mutagenesis. Mutation of both of these amino acids to the corresponding amino acids in 15-LOX-2 (Asp and Val, respectively) converted the positional specificity from 8S to 90% 15S without yielding any other by-products. Mutation of the corresponding residues in 15-LOX-2 to the 8-LOX sequence changed specificity to 50% oxygenation at C-8 for one amino acid substitution and 70% at C-8 for the double mutant. Based on the crystal structure of the reticulocyte 15-LOX, these two amino acids lie opposite the open coordination position of the catalytic iron in a likely site for substrate binding. The change from 8 to 15 specificity entails a switch in the head to tail binding of substrate. Enzymes that react with substrate "head first" (5-LOX and 8-LOX) have a bulky aromatic amino acid and a histidine in these positions, whereas lipoxygenases that accept substrates "tail first" (12-LOX and 15-LOX) have an aliphatic residue with a glutamine or aspartate. Thus, this positional determinant of the 8-LOX and 15-LOX-2 may have significance for other lipoxygenases.     

Control of prostaglandin stereochemistry at the 15-carbon by cyclooxygenases-1 and -2. A critical role for serine 530 and valine 349.

Prostaglandin synthesis by cyclooxygenases-1 and -2 (COX-1 and COX-2) involves an initial oxygenation of arachidonic acid at C-11, followed by endoperoxide and cyclopentane ring formation, and then a second reaction with molecular oxygen in the S configuration at C-15. The resulting 15S-hydroxyl group of prostaglandins is crucial for their bioactivity. Using human COX-1 and human and murine COX-2, we have identified two amino acids located in the oxygenase active site that control the stereochemistry at C-15. The most crucial determinant is Ser-530, the residue that is acetylated by aspirin. In COX-2, site-directed mutagenesis of Ser-530 to methionine, threonine, or valine produced highly active enzymes that formed 82-95% 15R-configuration prostaglandins; these have the opposite stereochemistry at C-15 to the natural products. In COX-1, the corresponding Ser-530 mutations inactivated the enzyme. The second residue, Val-349, exerts a more subtle influence. When Val-349 was replaced by isoleucine, the mutant COX-1 and COX-2 enzymes formed 41 and 65% 15R-prostaglandins, respectively. This change was highly specific for isoleucine, as mutations of Val-349 to alanine, leucine, asparagine, or threonine did not alter or only slightly altered (< or =13%) the S-configuration at C-15. These results establish a previously unrecognized role for Ser-530 and Val-349 in maintaining the correct S stereochemistry of the carbon-15 hydroxyl group during prostaglandin synthesis. The findings may also explain the absolute conservation of Ser-530, the target of aspirin, throughout the families of cyclooxygenase enzymes.     

Mutational and X-ray crystallographic analysis of the interaction of dihomo-gamma -linolenic acid with prostaglandin endoperoxide H synthases

Prostaglandin endoperoxide H synthases-1 and -2 (PGHSs) catalyze the committed step in prostaglandin biosynthesis. Both isozymes can oxygenate a variety of related polyunsaturated fatty acids. We report here the x-ray crystal structure of dihomo-gamma-linolenic acid (DHLA) in the cyclooxygenase site of PGHS-1 and the effects of active site substitutions on the oxygenation of DHLA, and we compare these results to those obtained previously with arachidonic acid (AA). DHLA is bound within the cyclooxygenase site in the same overall L-shaped conformation as AA. C-1 and C-11 through C-20 are in the same positions for both substrates, but the positions of C-2 through C-10 differ by up to 1.74 A. In general, substitutions of active site residues caused parallel changes in the oxygenation of both AA and DHLA. Two significant exceptions were Val-349 and Ser-530. A V349A substitution caused an 800-fold decrease in the V(max)/K(m) for DHLA but less than a 2-fold change with AA; kinetic evidence indicates that C-13 of DHLA is improperly positioned with respect to Tyr-385 in the V349A mutant thereby preventing efficient hydrogen abstraction. Val-349 contacts C-5 of DHLA and appears to serve as a structural bumper positioning the carboxyl half of DHLA, which, in turn, positions properly the omega-half of this substrate. A V349A substitution in PGHS-2 has similar, minor effects on the rates of oxygenation of AA and DHLA. Thus, Val-349 is a major determinant of substrate specificity for PGHS-1 but not for PGHS-2. Ser-530 also influences the substrate specificity of PGHS-1; an S530T substitution causes 40- and 750-fold decreases in oxygenation efficiencies for AA and DHLA, respectively.     

Alleles distribution of polymorphic promoter region C-590T in interleukin-4 genes and Q-576r and Ile-50Val regions in IL-4 receptor gene IL-4RA in patients with HCV-infection

81 patients with confirmed HCV-infection and 48 healthy volunteers were examined. In healthy Caucasian participants living in Siberian region significant predominance of C/T genotype in promoter region C-590T of interleukin-4 (IL-4) gene and Q/Q and Ile/Val genotypes in points -50 and -576 of IL-4RA gene that codes alpha-chains of IL-4 receptor were revealed. In patients with HCV-infection predominance of C/T genotype in C-590T region in IL-4 gene (OR = 1.86), R/R genotype in Q-576R region of IL-4RA gene (OR=7.86), and Val/Val genotype in point Ile-50Val (OR = 2.6) of the same gene. Summary predictive coefficient of hepatitis C development in carriers of these genotypes approached to 95%. During analysis of role of allelic polymorphism of IL-4 genes in predisposition to hepatitis C development it is necessary to consider not only presence of allelic variants of promoter regions of the IL-4 genes, but also the polymorphism of genes coding molecules binding with this cytokine on target cells membranes and in its soluble form.     

Characterization of free radicals formed from COX-catalyzed DGLA peroxidation

Like arachidonic acid (AA), dihomo-γ-linolenic acid (DGLA) is a 20-carbon ω-6 polyunsaturated fatty acid and a substrate of cyclooxygenase (COX). Through free radical reactions, COX metabolizes DGLA and AA to form well-known bioactive metabolites, namely, the 1 and 2 series of prostaglandins (PGs1 and PGs2), respectively. Unlike PGs2, which are viewed as proinflammatory, PGs1 possess anti-inflammatory and anticancer activities. However, the mechanisms linking the PGs to their bioactivities are still unclear, and radicals generated in COX-DGLA have not been detected. To better understand PG biology and determine whether different reactions occur in COX-DGLA and COX-AA, we have used LC/ESR/MS with a spin trap, α-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (POBN), to characterize the carbon-centered radicals formed from COX-DGLA in vitro, including cellular peroxidation. A total of five types of DGLA-derived radicals were characterized as POBN adducts: m/z 266, m/z 296, and m/z 550 (same as or similar to COX-AA) and m/z 324 and m/z 354 (exclusively from COX-DGLA). Our results suggest that C-15 oxygenation to form PGGs occurs in both COX-DGLA and COX-AA; however, C-8 oxygenation occurs exclusively in COX-DGLA. This new finding will be further investigated for its association with various bioactivities of PGs, with potential implications for inflammatory diseases.     

Sequence determinants for the reaction specificity of murine (12R)-lipoxygenase: targeted substrate modification and site-directed mutagenesis

Mammalian lipoxygenases (LOXs) are categorized with respect to their positional specificity of arachidonic acid oxygenation. Site-directed mutagenesis identified sequence determinants for the positional specificity of these enzymes, and a critical amino acid for the stereoselectivity was recently discovered. To search for sequence determinants of murine (12R)-LOX, we carried out multiple amino acid sequence alignments and found that Phe(390), Gly(441), Ala(455), and Val(631) align with previously identified positional determinants of S-LOX isoforms. Multiple site-directed mutagenesis studies on Phe(390) and Ala(455) did not induce specific alterations in the reaction specificity, but yielded enzyme species with reduced specific activities and stereo random product patterns. Mutation of Gly(441) to Ala, which caused drastic alterations in the reaction specificity of other LOX isoforms, failed to induce major alterations in the positional specificity of mouse (12R)-LOX, but markedly modified the enantioselectivity of the enzyme. When Val(631), which aligns with the positional determinant Ile(593) of rabbit 15-LOX, was mutated to a less space-filling residue (Ala or Gly), we obtained an enzyme species with augmented catalytic activity and specifically altered reaction characteristics (major formation of chiral (11R)-hydroxyeicosatetraenoic acid methyl ester). The importance of Val(631) for the stereo control of murine (12R)-LOX was confirmed with other substrates such as methyl linoleate and 20-hydroxyeicosatetraenoic acid methyl ester. These data identify Val(631) as the major sequence determinant for the specificity of murine (12R)-LOX. Furthermore, we conclude that substrate fatty acids may adopt different catalytically productive arrangements at the active site of murine (12R)-LOX and that each of these arrangements may lead to the formation of chiral oxygenation products.     

Structure-activity relationships in the dodecapeptide alpha-factor of Saccharomyces cerevisiae: position 6 analogues are poor inducers of agglutinability

Five des-Trp1,Cha3,X6 analogues of alpha-factor, where X = Ala, Val, Ile, Nle, or D-Leu and X = Leu in the natural alpha-factor sequence, were prepared by solution-phase techniques utilizing isobutyl chloroformate or 1-hydroxybenzotriazole accelerated active esters as the coupling agents. Purification to 98% or greater homogeneity was accomplished by high-performance liquid chromatography on a reversed-phase muBondapak C18 column with methanol/water/trifluoroacetic acid as the mobile phase. Three of the synthesized analogues (X6 = Val, Ile, Nle) induced morphogenesis and increased agglutinability in a cells. These substitutions demonstrate that a gamma-branched side chain at position 6 is not essential for biological activity. All of the active analogues induced morphogenesis at lower concentrations than they induced enhanced agglutinability. These results and other structure-activity relationships [Baffi, R. A., Shenbagamurthi, P., Terrance, K., Becker, J. M., Naider, F., & Lipke, P. (1984) J. Bacteriol. 158, 1152-1156] indicate that the agglutination and morphological responses to alpha-factor can be varied independently. Replacement of Leu6 with Ala or D-Leu resulted in inactive analogues that were not antagonistic for alpha-factor activity. Cell-mediated hydrolysis experiments indicated that the biological activities of the alpha-factor analogues are independent of their rates of degradation. All position 6 analogues were hydrolyzed more slowly than the parent compound, suggesting that the enzyme which degrades alpha-factor is highly specific for the native structure.     

pH dependence of 13C-15N coupling constants of highly 14N-enriched amino acids isolated from mass cultivation of algae.

The alga Ankistrodesmus braunii was grown with [14N]nitrate under optimized conditions of a large-scale mass cultivation. 19.7% of the dried algae were isolated as a mixture of amino acids. The 15N-labelled amino acids (15N content up to 98%) were separated by ion exchange chromatography using pyridine acetate gradients. The 15N cotent of the analytically pure amino acid was determined by combined gas-liquid chromatography-mass spectrometry of the trifluoroacetylated methylesters and by emission spectroscopy in the 15N analysator. Using pulse Fourier transform 13C nuclear magnetic resonance, the pH dependence of the 13C-15N coupling constants of Asp, Pro, Ser, Glu, Gly, Ala, Val, Ile and Leu was determined in aqueous solutions. Increasing coupling constants were found with pH and decreasing electron density, respectively. The relation of Binsch et al. (Binsch, G., Lambert, J.B., Roberts, B.W. and Roberts, J.D. (1964) J.Am. Chem. Soc. 86,5564-5570) between the coupling constant and the product of the S-part of the 13C and 15N hybridization SC - SN = 80 - J (13C-45X) fits best in acidic medium. The magnitude of coupling constants correlates well with the electron densities calculated by Del Re et al. (Del Re, G., Pullman, B. and Yonezawa, T. (1963) Biochim. Biophys. Acta 75, 153-182). The recording of 13C nuclear magnetic resonance spectra over the entire pH range revealed no change in the sign of the 13C-15N coupling constants of the amino acids.     

Shape and specificity in mammalian 15-lipoxygenase active site. The functional interplay of sequence determinants for the reaction specificity

Previous mutagenesis studies along with molecular modeling using the x-ray coordinates of the rabbit 15-lipoxygenase have led to the suggestion that the size of the substrate binding pocket may play an essential role in determining the oxygenation specificity of 5-, 12-, and 15-lipoxygenases. Based on the x-ray crystal structure of rabbit 15-lipoxygenase, Ile(593) appeared to be important in defining size and shape of the substrate-binding site in 15-lipoxygenases. We found that substitution of Ile(593) with alanine shifted the positional specificity of this enzyme toward 12-lipoxygenation. To compare the importance of position 593 with previously defined determinants for the oxygenation specificity, we introduced small (alanine-scan) or large amino acids (phenylalanine-scan) at critical positions surrounding the putative fatty acid-binding site, so that the volume of the pocket was either increased or decreased. Enlargement or alteration in packing density within the substrate binding pocket in the rabbit 15-lipoxygenase increased the share of 12-lipoxygenase products, whereas a smaller active site favored 15-lipoxygenation. Simultaneous substitution of both large and small residues in the context of either a 15- or 12-lipoxygenase indicated that there is a functional interplay of the sequence determinants for lipoxygenation specificity. If the 15-lipoxygenase active site is enlarged excessively, however, no lipoxygenation was observed anymore. Together these results indicate the importance of the overall size and shape of the arachidonic acid binding pocket in defining the specificity of lipoxygenase reaction.     

Insights from the X-ray crystal structure of coral 8R-lipoxygenase: calcium activation via a C2-like domain and a structural basis of product chirality

Lipoxygenases (LOXs) catalyze the regio- and stereospecific dioxygenation of polyunsaturated membrane-embedded fatty acids. We report here the 3.2 A resolution structure of 8R-LOX from the Caribbean sea whip coral Plexaura homomalla, a LOX isozyme with calcium dependence and the uncommon R chiral stereospecificity. Structural and spectroscopic analyses demonstrated calcium binding in a C2-like membrane-binding domain, illuminating the function of similar amino acids in calcium-activated mammalian 5-LOX, the key enzyme in the pathway to the pro-inflammatory leukotrienes. Mutation of Ca(2+)-ligating amino acids in 8R-LOX resulted not only in a diminished capacity to bind membranes, as monitored by fluorescence resonance energy transfer, but also in an associated loss of Ca(2+)-regulated enzyme activity. Moreover, a structural basis for R chiral specificity is also revealed; creation of a small oxygen pocket next to Gly(428) (Ala in all S-LOX isozymes) promoted C-8 oxygenation with R chirality on the activated fatty acid substrate.     

Investigation of the allene oxide pathway in the coral Plexaura homomalla: formation of novel ketols and isomers of prostaglandin A2 from 15-hydroxyeicosatetraenoic acid.

Prostaglandin A2 is a major constituent of the gorgonian Plexaura homomalla, and there is evidence that its biosynthesis involves a noncyclooxygenase pathway. The coral contains an 8(R)-lipoxygenase and an allene oxide synthase; from arachidonic acid, the sequential action of these enzymes gives an allene epoxide, the cyclization of which forms an analogue of prostaglandin A2 (PGA2) with no 15-hydroxyl group. In this study we examined the metabolic fate of 15-hydroxyeicosatetraenoic acid (15-HETE), which via analogous reactions could lead to PGA2. The 8(R)-lipoxygenase metabolized preferentially the 15(R) enantiomer of 15-HETE, and this reaction was stimulated fivefold by including 1 M NaCl in the incubation. Further enzymic steps were detected by comparing the metabolic profiles of the 8(R)-hydroperoxy-15(R)-hydroxy intermediate with that of its 8(S),15(S) enantiomer. Two main products were formed exclusively from the 8(R),15(R) enantiomer: an allene epoxide and the comparatively stable epoxide, 8,9-epoxy-10,15-dihydroxyeicosa-5,11,14-trienoic acid. Formation of the allene oxide was inferred from detection of its hydrolysis and cyclization products. It cyclized to give two isomers of PGA2 which have a "cis" arrangement of the side chains. The main hydrolysis product (8,15-dihydroxy-9-ketoeicosa-5,11,13-trienoic acid) was unstable and prone to oxygenation, giving 8,14,15-trihydroxy-9-ketoeicosa-5,10,12-trienoic acids after reduction of the 14-hydroperoxide. We conclude that metabolism of a 15-hydroxy eicosanoid is a potential route to the A series prostaglandins, although the low yield and lack of stereochemical control suggest that this is not the natural pathway of biosynthesis in P. homomalla. Unexpectedly, the major end products of the pathway are trihydroxy ketols and the single diastereomer of a stable epoxyalcohol.     

Extended substrate specificity of rat mast cell protease 5, a rodent alpha-chymase with elastase-like primary specificity

Chymases are mast cell serine proteases with chymotrypsin-like primary substrate specificity. Amino acid sequence comparisons of alpha-chymases from different species indicated that certain rodent alpha-chymases have a restricted S1 pocket that could only accommodate small amino acids, i.e. they may, despite being classified as chymases, in fact display elastase-like substrate specificity. To explore this possibility, the alpha-chymase, rat mast cell protease 5 (rMCP-5), was produced as a proenzyme with a His6 purification tag and an enterokinase-susceptible peptide replacing the natural propeptide. After removal of the purification tag/enterokinase site by enterokinase digestion, rMCP-5 bound the serine-protease-specific inhibitor diisopropyl fluorophosphate, showing that rMCP-5 was catalytically active. The primary specificity was investigated with chromogenic substrates of the general sequence succinyl-Ala-Ala-Pro-X-p-nitroanilide, where the X was Ile, Val, Ala, Phe or Leu. The activity was highest toward substrates with Val or Ala in the P1 position, whereas low activity toward the peptide with a P1 Phe was observed, indicating that the substrate specificity of rMCP-5 indeed is elastase-like. The extended substrate specificity was examined utilizing a phage-displayed random nonapeptide library. The preferred cleavage sequence was resolved as P4-(Gly/Pro/Val), P3-(Leu/Val/Glu), P2-(Leu/Val/Thr), P1-(Val/Ala/Ile), P1'-(Xaa), and P2'-(Glu/Leu/Asp). Hence, the extended substrate specificity is similar to human chymase in most positions except for the P1 position. We conclude that the rat alpha-chymase has converted to elastase-like substrate specificity, perhaps associated with an adoption of new biological targets, separate from those of human alpha-chymase.     

Hematopoietic prostaglandin D synthase (HPGDS): a high stability, Val187Ile isoenzyme common among African Americans and its relationship to risk for colorectal cancer

Intestinal tumors in Apc(Min/+) mice are suppressed by over-production of HPGDS, which is a glutathione transferase that forms prostaglandin D(2) (PGD(2)). We characterized naturally occurring HPGDS isoenzymes, to see if HPGDS variation is associated with human colorectal cancer risk. We used DNA heteroduplex analysis and sequencing to identify HPGDS variants among healthy individuals. HPGDS isoenzymes were produced in bacteria, and their catalytic activities were tested. To determine in vivo effects, we conducted pooled case-control analyses to assess whether there is an association of the isoenzyme with colorectal cancer. Roughly 8% of African Americans and 2% of Caucasians had a highly stable Val187lle isoenzyme (with isoleucine instead of valine at position 187). At 37°C, the wild-type enzyme lost 15% of its activity in 1h, whereas the Val187Ile form remained >95% active. At 50°C, the half life of native HPGDS was 9min, compared to 42 min for Val187Ile. The odds ratio for colorectal cancer among African Americans with Val187Ile was 1.10 (95% CI, 0.75-1.62; 533 cases, 795 controls). Thus, the Val187Ile HPGDS isoenzyme common among African Americans is not associated with colorectal cancer risk. Other approaches will be needed to establish a role for HPGDS in occurrence of human intestinal tumors, as indicated by a mouse model.     

Genetic variants of the receptors for thromboxane A2 and IL-4 in atopic dermatitis

Thromboxane A2 (TXA2) is an arachidonate metabolite which is considered to relate to chronic inflammation in atopic diseases characterized by elevated immunoglobulin E productivity. The elevation of immunoglobulin E levels involves many molecules including interleukin-4 (IL-4) and interleukin-4 receptor alpha chain (IL-4R alpha). To assess whether genetic variants of TXA2 receptor, IL-4 and IL-4R alpha genes relate to the elevation of serum immunoglobulin E levels in patients with atopic dermatitis (AD), we conducted an association study of genetic polymorphisms of TXA2 receptor (795C/T), IL-4 (-589C/T), and IL-4R alpha (Ile50Val) in a Japanese population (n = 789). The TXA2 receptor 795TT genotype strongly related to AD with high serum immunoglobulin E concentrations. AD patients with both TXA2 receptor 795TT genotype and the IL-4R alpha Ile50/Ile50 genotype showed the greatest immunoglobulin E concentrations. These results suggest TXA2 receptor polymorphism strongly interacts with IL-4R alpha polymorphism as a major determinant of high serum immunoglobulin E levels in AD.     

Role of residue 478 as a determinant of the substrate specificity of cytochrome P450 2B1.

Two allelic variants and eight site-directed mutants of cytochrome P450 2B1 differing at residue 478 have been expressed in COS cells and assayed for androstenedione hydroxylase activities. The 478Gly and 478Ala variants and five mutants (Ser, Thr, Val, Ile, and Leu) exhibited 16 beta-OH:16 alpha-OH ratios ranging from 0.7 to 9.3, whereas the Pro, Glu, and Arg mutants were expressed but inactive. The seven samples active toward androstenedione also exhibited testosterone 16 beta-OH:16 alpha-OH ratios ranging from 0.4 to 2.3. With both steroids, the Gly variant had the highest 16 beta-hydroxylase activity, and the 16 beta-OH:16 alpha-OH ratio increased with the size of aliphatic size chains (Ala, Val, and Ile/Leu). The highest ratio of androgen 15 alpha:16-hydroxylation was observed with the Ser mutant. On the basis of previous work indicating decreased susceptibility of the 478Ala variant in liver microsomal and reconstituted systems to inactivation by chloramphenicol analogs, methodology was refined for monitoring enzyme inactivation in COS cell microsomes. The Gly and Ala variants were inactivated by chloramphenicol with similar rate constants, whereas the Ser and Val mutants were inactivated more slowly, and the Leu mutant was refractory. Only the Gly variant was inactivated by the chloramphenicol analog N-(2-p-nitrophenethyl)chlorofluoroacetamide. Thus, the side chain of residue 478 appears to be a major determinant of enzyme inactivation as well as of androgen hydroxylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure of arachidonic acid bound to a mutant of prostaglandin endoperoxide H synthase-1 that forms predominantly 11-hydroperoxyeicosatetraenoic acid

Kinetic studies and analysis of the products formed by native and mutant forms of ovine prostaglandin endoperoxide H synthase-1 (oPGHS-1) have suggested that arachidonic acid (AA) can exist in the cyclooxygenase active site of the enzyme in three different, catalytically competent conformations that lead to prostaglandin G2 (PGG2), 11R-hydroperoxyeicosatetraenoic acid (HPETE), and 15R,S-HPETE, respectively. We have identified an oPGHS-1 mutant (V349A/W387F) that forms predominantly 11R-HPETE. Thus, the preferred catalytically competent arrangement of AA in the cyclooxygenase site of this double mutant must be one that leads to 11-HPETE. The crystal structure of Co3+-protoporphyrin IX V349A/W387F oPGHS-1 in a complex with AA was determined to 3.1 A. Significant differences are observed in the positions of atoms C-3, C-4, C-5, C-6, C-10, C-11, and C-12 of bound AA between native and V349A/W387F oPGHS-1; in comparison, the positions of the side chains of cyclooxygenase active site residues are unchanged. The structure of the double mutant presented here provides structural insight as to how Val349 and Trp387 help position C-9 and C-11 of AA so that the incipient 11-peroxyl radical intermediate is able to add to C-9 to form the 9,11 endoperoxide group of PGG2. In the V349A/W387F oPGHS-1.AA complex the locations of C-9 and C-11 of AA with respect to one another make it difficult to form the endoperoxide group from the 11-hydroperoxyl radical. Therefore, the reaction apparently aborts yielding 11R-HPETE instead of PGG2. In addition, the observed differences in the positions of carbon atoms of AA bound to this mutant provides indirect support for the concept that the conformer of AA shown previously to be bound within the cyclooxygenase active site of native oPGHS-1 is the one that leads to PGG2.     

Genetic polymorphism of the glutathione-S-transferase P1 gene (GSTP1) and susceptibility to prostate cancer in the Kashmiri population

Glutathione-S-transferase P1 (GSTP1) is a critical enzyme of the phase II detoxification pathway. One of the common functional polymorphisms of GSTP1 is A→G at nucleotide 313, which results in an amino acid substitution (Ile105Val) at the substrate binding site of GSTP1 and reduces catalytic activity of GSTP1. To investigate the GSTP1 Ile105Val genotype frequency in prostate cancer cases in the Kashmiri population, we designed a case-control study, in which 50 prostate cancer cases and 45 benign prostate hyperplasia cases were studied for GSTP1 Ile105Val polymorphism, compared to 80 controls taken from the general population, employing the PCR-RFLP technique. We found the frequency of the three different genotypes of GSTP1 Ile105Val in our ethnic Kashmir population, i.e., Ile/Ile, Ile/Val and Val/Val, to be 52.4, 33.3 and 14.3% among prostate cancer cases, 48.5, 37.5 and 14% among benign prostate hyperplasia cases and 73.8, 21.3 and 5% in the control population, respectively. There was a significant association between the GSTP1 Ile/Val genotype and the advanced age group among the cases. We conclude that GSTP1 Ile/Val polymorphism is involved in the risk of prostate cancer development in our population.