Three-dimensional structure of rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase: a member of the aldo-keto reductase superfamily.

F series prostaglandins (PG)1 are widely distributed in various organs of mammals and exhibit a variety of biological activities including constriction of pulmonary arteries (1, 2). PGF 2 is synthesized from PGE 2 , PGD 2 , or PGH 2 by PGE 9-ketoreductase, PGD 11-ketoreductase, or PGH 9,11-endoperoxide reductase, respectively. PGF synthase (EC 1.1.1.188) was purified from bovine lung by Watanabe et al. (3). It forms 9␣,11␤-PGF 2 (4) from PGD 2 (PGD 2 11-ketoreductase activity) and PGF 2␣ from PGH 2 (PGH 2 9,11-endoperoxide reductase activity) on the same molecule in the presence of NADPH (3, 4). This enzyme catalyzes the reduction of other carbonyl compounds including 9,10-phenanthrenequinone (PQ) as well as that of PGD 2 and PGH 2 but does not catalyze the reduction of PGE 2 . Although PGD 2 11-ketoreductase activity was competitively inhibited by PQ, PGH 2 9,11-endoperoxide reductase activity was not inhibited by PGD 2 or PQ (3). PGF synthase belongs to the aldo-keto reductase family. The bovine lung PGF synthase is a monomeric protein with a M r of 36,666 consisting of 323 amino acids, and its amino acid sequence shows high homology compared with that of other aldo-keto reductase family members (5). PGF synthase has two isozymes, one in the lung (3) and the other one in the liver (6), with different K m values for PGD 2 (120 and 10 M, respectively). The regulation by metals, the sensitivity to chloride ions, the inhibition by CuSO 4 and HgCl 2 , and the profile of immuno-precipitation with anti-bovine lung PGF synthase antibody are different between the two isozymes (6). Although Kuchinke et al. (7) isolated a clone (PGFS II) of PGF synthase from bovine liver and determined its amino acid sequence, the 99% similarity with the amino acid sequence of lung PGF synthase and the high K m value for PGD 2 of this recombinant PGFS II indicated that its cDNA was that of the lung-type enzyme even though it had been isolated from liver. Until now, the primary structure of the liver-type PGF synthase and the amino acids related to the affinity for PGD 2

Section: Discussionmentioning

confidence: 99%

cDNA Cloning, Expression, and Mutagenesis Study of Liver-type Prostaglandin F Synthase

Suzuki

Fujii

Miyano

et al. 1999

“…They catalyze the 4-pro-R hydride transfer from NADPH to the acceptor carbonyl on the steroid substrate in an ordered bi-bi reaction where NAD(P)(H) binds first and leaves last (16,17). Rat liver 3␣-HSD (AKR1C9), which shares 69% sequence identity with its human homologs, represents the best system to relate structure to function for HSDs, because crystal structures exist for the apoenzyme (E) (18), the E⅐NADP ϩ binary complex (19), and the E⅐NADP ϩ ⅐testosterone ternary complex (where testosterone is a competitive inhibitor) (20). These "snapshots" of the enzyme along the reaction pathway reveal that significant conformational changes occur upon the binding of each ligand and provide a structural basis for the ordered bi-bi mechanism (Fig.…”

mentioning

confidence: 99%

Elucidation of a Complete Kinetic Mechanism for a Mammalian Hydroxysteroid Dehydrogenase (HSD) and Identification of All Enzyme Forms on the Reaction Coordinate

Cooper

Jin

Penning

2007

Hydroxysteroid dehydrogenases (HSDs) are essential for the biosynthesis and mechanism of action of all steroid hormones. We report the complete kinetic mechanism of a mammalian HSD using rat 3␣-HSD of the aldo-keto reductase superfamily (AKR1C9) with the substrate pairs androstane-3,17-dione and NADPH (reduction) and androsterone and NADP ؉ (oxidation). Steady-state, transient state kinetics, and kinetic isotope effects reconciled the ordered bi-bi mechanism, which contained 9 enzyme forms and permitted the estimation of 16 kinetic constants. In both reactions, loose association of the NADP(H) was followed by two conformational changes, which increased cofactor affinity by >86-fold. For androstane-3,17-dione reduction, the release of NADP ؉ controlled k cat , whereas the chemical event also contributed to this term. k cat was insensitive to [ 2 H]NADPH, whereas D k cat /K m and the D k lim (ratio of the maximum rates of single turnover) were 1.06 and 2.06, respectively. Under multiple turnover conditions partial burst kinetics were observed. For androsterone oxidation, the rate of NADPH release dominated k cat , whereas the rates of the chemical event and the release of androstane-3,17-dione were 50-fold greater. Under multiple turnover conditions full burst kinetics were observed. Although the internal equilibrium constant favored oxidation, the overall K eq favored reduction. The kinetic Haldane and free energy diagram confirmed that K eq was governed by ligand binding terms that favored the reduction reactants. Thus, HSDs in the aldo-keto reductase superfamily thermodynamically favor ketosteroid reduction. Mammalian hydroxysteroid dehydrogenases (HSDs)3 play pivotal roles in steroid hormone biosynthesis and metabolism (1, 2). In target tissues, they regulate the amount of steroid hormone available for their cognate nuclear receptor (3-7). This is achieved by the positional and stereospecific oxidoreduction of potent steroid hormones to their corresponding inactive metabolites or vice versa. Often these interconversions are catalyzed by pairs of HSDs that function preferentially as reductases or oxidases (5-8). There are two superfamilies of HSDs, the soluble aldo-keto reductase (AKRs) (9) and the mainly membrane-associated shortchain dehydrogenase reductases (SDRs) (10). Several HSDs have become drug targets to manipulate steroid hormone action in target tissues (11-13).Four human HSDs from the AKR superfamily, AKR1C1-AKR1C4, can reduce 3-, 17-, and 20-ketosteroids in different ratios (14, 15). They catalyze the 4-pro-R hydride transfer from NADPH to the acceptor carbonyl on the steroid substrate in an ordered bi-bi reaction where NAD(P)(H) binds first and leaves last (16,17). Rat liver 3␣-HSD (AKR1C9), which shares 69% sequence identity with its human homologs, represents the best system to relate structure to function for HSDs, because crystal structures exist for the apoenzyme (E) (18), the E⅐NADP ϩ binary complex (19), and the E⅐NADP ϩ ⅐testosterone ternary complex (where testosterone is a competitive inh...

“…2B). The number of interactions is lower than that for AKRs of the first subfamily, such as human ADR holoenzyme and rat 3␣-HSD, both of which have 19 hydrogen bonds, 3 salt bridges, and 1 aromatic stacking (2,4). Fluorescence quenching assay (data not shown) revealed that the K d value of TbPGFS for NADPH (5.3 M) was 1 order of magnitude higher than that of rat 3␣-HSD, i.e.…”

Section: Resultsmentioning

confidence: 99%

“…Histidine facilitates proton donation during reduction (46) or orientates the substrate carbonyl in the active site (9,43), whereas lysine helps proton removal by tyrosine during oxidation by making a hydrogen bond that lowers the pK a value of the tyrosine. Aspartate forms a salt bridge to stabilize lysine (3,4,40). In order to catalyze the oxidation/reduction reaction according to this mechanism, tyrosine OH has been involved in hydrogen bond networks with histidine and lysine.…”

Section: Figmentioning

confidence: 99%

Structural and Mutational Analysis of Trypanosoma brucei Prostaglandin H2 Reductase Provides Insight into the Catalytic Mechanism of Aldo-ketoreductases

Kilunga

Inoue

Okano³

et al. 2005

Trypanosoma brucei prostaglandin F 2␣ synthase is an aldo-ketoreductase that catalyzes the reduction of prostaglandin H 2 to PGF 2␣ in addition to that of 9,10-phenanthrenequinone. We report the crystal structure of TbPGFS⅐NADP protonation through a water molecule, while exerting an electrostatic repulsion against His 110 that maintains the spatial arrangement which allows the formation of a hydrogen bond between His 110 and C 11 carbonyl of PGH 2 . We also show that Tyr 52 acts as the general acid catalyst for 9,10-PQ reduction, and thus we not only elucidate the catalytic mechanism of a PGH 2 reductase but also provide an insight into the catalytic specificity of AKRs.