Structural Basis of Leukotriene B4 12-Hydroxydehydrogenase/15-Oxo-prostaglandin 13-Reductase Catalytic Mechanism and a Possible Src Homology 3 Domain Binding Loop

Abstract

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 -chain of 15-oxo-PGE 2 was defined in the electron density map in the ternary complex structure. The -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 prolinerich helix at the dimer interface and regulate LTB 4 12-HD/PGR activity by disruption of the substrate binding pore to accommodate the -chain.Eicosanoids, such as prostaglandins, leukotrienes, and lipoxins, are endogenous lipid mediators and play key roles in a wide range of normal physiological and pathophysiological processes such as host defense responses and inflammation. The dominant eicosanoids, leukotriene B 4 (LTB 4 ), 1 prostaglandin E and F (PGE and PGF), and lipoxin A 4 (LXA 4 ) are rapidly inactivated by enzymatic degradation. A bifunctional LTB 4 12-hydroxydehydrogenase/15-oxo-prostaglandin 13-reductase (LTB 4 12-HD/PGR), a member of the zinc-independent medium chain dehydrogenase/reductase (MDR) family (1), is a key enzyme in the irreversible degradation of all three eicosanoids to remove these highly biologically active agents (2-4) (Fig. 1).PGE 2 mediates a wide range of physiological processes in a variety of tissues (Fig. 1). It is involved in the relaxation of smooth muscle cells to protect against lung inflammation (5), regulating gastric acid and mucus secretion in the stomach (6), regulating water and mineral balance in the kidney (7), as well as having roles in immune responses, thermo-regulation, and vasopermeability. Overproduction of PGE 2 induces inflammation and can have oncogenic effects (8). PGE 2 production can be inhibited by nonsteroidal anti-inflammatory drugs (NSAIDs), which act as cyclooxygenase inhibitors. NSAIDs are important drugs in the treat...

Mechanistic and Structural Studies of Apoform, Binary, and Ternary Complexes of the Arabidopsis Alkenal Double Bond Reductase At5g16970

“…As discussed earlier, the latter catalyzes the reduction of a very broad array of ␣,␤-unsaturated ketones and aldehydes, including toxic products of lipid peroxidation in addition to that of the 13-14-double bond of 15-oxoprostaglandins (29,56). Accordingly, most of the residues constituting the NADP(H)-binding sites are very similar between AtDBR1 and 12-HD/PGR.…”

“…For guinea pig 12-HD/PGR and rat liver AOR, a ketoreductase reaction mechanism has been proposed with a conjugated enolate as the reaction intermediate (29,55). Apparently, the latter enzyme is also able to reduce one of the phenylpropanoids, cinnamyl aldehyde (17) (30), whereas AtDBR1 does not reduce it.…”

“…Crystals of the other ternary complex (AtDBR1-NADP ϩ -4-HNE (15): ternary II) were produced by soaking the binary complex crystal in a solution of 1 mM 4-HNE (15) (Alexis Biochemicals Inc.). All binary and ternary complexes crystallized in an orthorhombic space group, P2 1 Structural Solution and Refinement-The structure of apoAtDBR1 was solved by the molecular replacement method using a coordinate of the guinea pig (Cavia porcellus) leukotriene B 4 12-HD/PGR (PDB code 1V3V) (29) and the software package AMoRe (48). The rigid body refinement of the initial position was carried out by using 15.0 -3.0 Å resolution data and gave an R-value of 35%.…”

“…Crystals of both ternary complexes were obtained by mixing AtDBR1 with NADP ϩ /p-coumaryl aldehyde (6) and by soaking the AtDBR1/NADP ϩ binary complex crystals in a solution containing 4-HNE (15), respectively. The apoform of AtDBR1 was determined at 2.5 Å resolution by the molecular replacement method using coordinates of the aforementioned 12-HD/PGR (PDB code 1V3V) from guinea pig (C. porcellus) (29), which shows 56/41% similarity/identity to AtDBR1 (Table 1). Additionally, the binary and ternary complex structures of AtDBR1 were determined at 2.8 Å resolution using the coordinates of the deduced structure of the AtDBR1 apoform.…”

“…2C and Fig. 3) (29), and in rat liver detoxification of 4-hydroxy-(2E)-nonenal (4-HNE (15)) (Figs. 2D and 3) (30).…”