Leukotriene B4 12-hydroxydehydrogenase/15-ketoprostaglandin Delta 13-reductase (LTB4 12-HD/PGR) responsible for the reduction of a double-bond of the alpha,beta-unsaturated ketone of an aryl propionic acid non-steroidal anti-inflammatory agent CS-670.

CS-670 is a non-steroidal anti-inflammatory agent with an alpha,beta-unsaturated ketone structure. It exerts its pharmacological activity after being transformed to the active metabolite (2S,1'R,2'S)-trans-alcohol. Two consecutive reductions are needed for the formation of the active metabolite, reduction of the double-bond of the alpha,beta-unsaturated ketone moiety, followed by reduction of the resulting saturated ketone. The objective of the current study was to identify the enzyme responsible for reduction of the double-bond. An enzyme purified from rat liver cytosol as a single band on sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was analysed by a Mascot database search of nano-LC tandem mass spectrometry (MS/MS) data and the enzyme was identified as 2-alkenal reductase (EC 1.3.1.74), which is known as an beta-nicotinamide adenine dinucleotide phosphate (NADPH)-dependent alkenal/one oxidoreductase and has a role for leukotriene B(4) 12-hydroxydehydrogenase/15-ketoprostaglandinDelta13-reductase (LTB(4) 12-HD/PGR). The identification was confirmed by cloning LTB(4) 12-HD/PGR cDNA from rat liver, expressing it in Escherichia coli, and characterizing the properties of the enzyme. The identity was further supported by the subcellular localization in cytosol, a cofactor requirement for NADPH, substrate specificity, and substantial inhibition by 15-ketoPGF(2alpha), benzylideneacetophenone, indomethacin, and quercitrin. In addition to catalysing the biological reduction of eicosanoids, including prostaglandins, leukotrienes, and lipoxins, LTB(4) 12-HD/PGR was also determined to function as a xenobiotic-metabolizing enzyme.

Crystal structure of anti-configuration of indomethacin and leukotriene B4 12-hydroxydehydrogenase/15-oxo-prostaglandin 13-reductase complex reveals the structural basis of broad spectrum indomethacin efficacy.

The crystal structure of the ternary complex of leukotriene B4 12-hydroxydehydrogenase/15-oxo-prostaglandin (15-oxo-PG) 13-reductase (LTB4 12HD/PGR), an essential enzyme for eicosanoid inactivation pathways, with indomethacin and NADP+ has been solved. An indomethacin molecule bound in the anti-configuration at one of the two active site clefts of the homo-dimer interface in the LTB4 12HD/PGR and was confirmed by a binding calorimetry. The chlorobenzene ring is buried in the hydrophobic pore used as a binding site by the omega-chain of 15-oxo-PGE2. The carboxyl group interacts with the guanidino group of Arg56 and the phenolic hydroxyl group of Tyr262. Indomethacin shows a broad spectrum of efficacy against lipid-mediator related proteins including cyclooxygenase-2, phospholipase A2, PGF synthase and PGE synthase-2 but in the syn-configuration as well as LTB4 12HD/PGR in the anti-configuration. Indomethacin does not necessarily mimic the binding mode of the lipid-mediator substrates in the active sites of these complex structures. Thus, the broad spectrum of indomethacin efficacy can be attributed to its ability to adopt a range of different stable conformations. This allows the indomethacin to adapt to the distinct binding site features of each protein whilst maintaining favorable interactions between the carboxyl group and a counter charged functional group.

Structural basis of leukotriene B4 12-hydroxydehydrogenase/15-Oxo-prostaglandin 13-reductase catalytic mechanism and a possible Src homology 3 domain binding loop.

The bifunctional leukotriene B(4) 12-hydroxydehydrogenase/15-oxo-prostaglandin 13-reductase (LTB(4) 12-HD/PGR) is an essential enzyme for eicosanoid inactivation. It is involved in the metabolism of the E and F series of 15-oxo-prostaglandins (15-oxo-PGs), leukotriene B(4) (LTB(4)), and 15-oxo-lipoxin A(4) (15-oxo-LXA(4)). Some nonsteroidal anti-inflammatory drugs (NSAIDs), which primarily act as cyclooxygenase inhibitors also inhibit LTB(4) 12-HD/PGR activity. Here we report the crystal structure of the LTB(4) 12-HD/PGR, the binary complex structure with NADP(+), and the ternary complex structure with NADP(+) and 15-oxo-PGE(2). In the ternary complex, both in the crystalline form and in solution, the enolate anion intermediate accumulates as a brown chromophore. PGE(2) contains two chains, but only the omega-chain of 15-oxo-PGE(2) was defined in the electron density map in the ternary complex structure. The omega-chain was identified at the hydrophobic pore on the dimer interface. The structure showed that the 15-oxo group forms hydrogen bonds with the 2'-hydroxyl group of nicotine amide ribose of NADP(+) and a bound water molecule to stabilize the enolate intermediate during the reductase reaction. The electron-deficient C13 atom of the conjugated enolate may be directly attacked by a hydride from the NADPH nicotine amide in a stereospecific manner. The moderate recognition of 15-oxo-PGE(2) is consistent with a broad substrate specificity of LTB(4) 12-HD/PGR. The structure also implies that a Src homology domain 3 may interact with the left-handed proline-rich helix at the dimer interface and regulate LTB(4) 12-HD/PGR activity by disruption of the substrate binding pore to accommodate the omega-chain.

Multiplicity of rat liver 15-ketoprostaglandin delta 13-reductases.

Five kinds of 15-keto-PG delta 13-reductases (enzymes I, II, III, IV and V) were separated and purified from rat liver cytosol. Four (enzymes I,II, III and IV) out of these enzymes were homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights of enzymes I, II, III and IV were estimated to be 40,000, 25,000, 64,000 and 70,000 by the electrophoresis, and 42,000, 23,000, 66,000 and 72,000 by gel filtration on a Sephadex G-200 column, respectively. All of these enzymes exhibited the NADPH-dependent activities. In the cases of enzymes I, III and V, NADH was also effective as an electron donor, but to a lesser extent in enzymes I and III. The apparent K(m) values of enzymes I, II, III, IV and V for 15-keto-PGF2 alpha with NADPH were 276, 875, 842, 948 and 2767 nM. The enzymes had isoelectric points at 4.5, 4.9, 6.2, 6.4 and 5.4, respectively. Enzyme I exhibited the double bond reductase activities toward alpha, beta-ketoalkenes such as trans-benzylidene-acetone and trans-phenyl-1-propenylketone. Enzymes III and IV also catalyzed the double bond reduction of trans-phenyl-1-propenyl-ketone. All of these enzymes were markedly inhibited by various chemicals such as dicumarol, quercitrin, p-chloromercuri-benzoic acid, 5,5'-dithio-bis(2-nitrobenzoic acid) and so on.

Purification of a 15-ketoprostaglandin delta 13-reductase from rat liver and its ability to reduce the double bond of xenobiotics.

A 15-ketoprostaglandin delta 13-reductase was purified to homogeneity from rat liver. The enzyme used NADPH much more effectively than NADH as an electron donor. The molecular weight was estimated to be 39,500 by electrophoresis and 42,000 by gel filtration. The Km apparent for 15-ketoprostaglandin F2 alpha was 213 nM. The enzyme was markedly inhibited by dicumarol, quercitrin, p-chloromercuribenzoic acid and indomethacin. The enzyme had an isoelectric point at pH 4.5 and a broad pH optimum. The enzyme also exhibited the double bond reductase activity toward several xenobiotics with the double bond adjacent to the carbonyl group.