Mechanistic insights from the three-dimensional structure of 3-oxo-Delta(5)-steroid isomerase.

3-Oxo-Delta(5)-steroid isomerase (KSI) catalyzes the isomerization of beta,gamma-unsaturated 3-oxosteroids to their conjugated isomers through the formation of an intermediate dienolate. The three-dimensional structure of the enzyme from Pseudomonas testosteroni was solved by multidimensional heteronuclear magnetic resonance spectroscopy. This protein, a 28-kDa symmetric dimer, exhibits a three-dimensional fold with the two independently folded monomers packed together via extensive hydrophobic and electrostatic interactions. The previously identified catalytically important residues Tyr-14 (general acid) and Asp-38 (general base) are located near the bottom of a deep hydrophobic cavity and are positioned in a manner consistent with previous mechanistic hypotheses. The structure also revealed the presence of an unexpected acid group (Asp-99) located in the active site adjacent to Tyr-14. Mutagenesis and kinetic studies show that Asp-99 has an anomalously high pK(a) (>9), which allows it to contribute to catalysis by donating a hydrogen bond to the intermediate and to the transition states. In support of this hypothesis, effects on the kinetic parameters of the mutations Y14F and D99A are additive in the Y14F/D99A mutant.

Electrophilic assistance by Asp-99 of 3-oxo-Delta 5-steroid isomerase.

3-Oxo-Delta 5-steroid isomerase (Delta 5-3-ketosteroid isomerase, KSI; EC 5.3.3.1) catalyzes the conversion of a variety of beta, gamma-unsaturated 3-oxosteroids to their corresponding alpha, beta-unsaturated isomers at rates that approach the diffusion limit for specific substrates. The reaction proceeds through a dienolate intermediate, with two amino acid residues (Asp-38 and Tyr-14) known to be involved in catalysis. When the complete three-dimensional structure of KSI was determined recently by NMR methods, an additional polar residue (Asp-99) was found in the active site and this group was shown to be important for catalytic activity. In this work, we examine the properties of several mutant KSIs to determine the nature of catalysis by Asp-99 of KSI. The electrophoretic mobilities of wild-type (WT) KSI and several mutants (D99A, D99N, D38N, and D38N/D99A) on native gels were determined at pH values ranging from 6.0 to 8.5. The results demonstrate that the pKa of Asp-99 is >8.5 in wild-type KSI. The pH-rate profiles for the D99A, D99N, and D38H/D99A mutants of KSI were also determined. For all three mutants, kcat and kcat/KM do not decrease at high pH, in contrast to those for WT and D38H, which lose activity above pH 9 and 8, respectively. Mutation of Asp-99 to Asn decreases kcat for the substrate 5-androstene-3,17-dione by 27-fold and kcat/Km by 23-fold, substantially less than the loss of activity (3000-fold in kcat and 2200-fold in kcat/Km) observed when Asp-99 is mutated to Ala, consistent with a hydrogen bonding role for Asp-99. Taken together, these results provide evidence that Asp-99 participates in catalysis in its protonated form, with a pKa of >9 in WT and approximately 8.5 in the D38H mutant. Asp-99 likely donates a hydrogen bond to O-3 of the steroid, helping to stabilize the transition state(s) of the KSI-catalyzed reaction.

Identification of an intronic enhancer that nullifies upstream repression of SPARC gene expression.

The SPARC gene 5'flanking sequence has been shown to contain enhancer elements, but also negative control elements immediately upstream of the enhancer elements. Although these 5'enhancer elements are active in F9 and PYS-2 cells, their activities are nullified by the 5'repressor activity. In the present study we have identified within intron 1 between nucleotides (nt) +5000 and +5150 of the SPARC gene an enhancer element that bound to two transcription factors of 48 and 52 kDa and between nt +5000 and +5523 a DNase I hypersensitive site. Furthermore, a region containing the 3'intron 1 enhancer element, together with the 5'enhancer elements, neutralized the 5'repressor activity and stimulated efficient transcription. The resulting SPARC promoter activity is about equal in F9, differentiated F9 and PYS-2 cells. We consistently found that the rate of SPARC transcription is nearly the same in F9 and PYS-2 cells. Association of the 3'enhancer element in intron 1 with the DNase I hypersensitive site suggests that both play a role in regulating SPARC expression in vivo .

Solution structure of 3-oxo-delta5-steroid isomerase.

The three-dimensional structure of the enzyme 3-oxo-delta5-steroid isomerase (E.C. 5.3.3.1), a 28-kilodalton symmetrical dimer, was solved by multidimensional heteronuclear magnetic resonance spectroscopy. The two independently folded monomers pack together by means of extensive hydrophobic and electrostatic interactions. Each monomer comprises three alpha helices and a six-strand mixed beta-pleated sheet arranged to form a deep hydrophobic cavity. Catalytically important residues Tyr14 (general acid) and Asp38 (general base) are located near the bottom of the cavity and positioned as expected from mechanistic hypotheses. An unexpected acid group (Asp99) is also located in the active site adjacent to Tyr14, and kinetic and binding studies of the Asp99 to Ala mutant demonstrate that Asp99 contributes to catalysis by stabilizing the intermediate.

Mechanism-based inactivation of thymine hydroxylase, an alpha-ketoglutarate-dependent dioxygenase, by 5-ethynyluracil.

5-Ethynyluracil was shown to be a mechanism-based inactivator of thymine 7-hydroxylase, with Ki = 22 microM and a k2 = 2.6 min-1l Inactivation resulted in covalent modification of the enzyme with a stoichiometry of approximately 1 adduct/enzyme molecule. The reaction of thymine 7-hydroxylase with 5-ethynyluracil also generated two products: 5-carboxyuracil and uracil-5-acetylglycine. The enzyme adduct was stable at pH 2, 8, and 10 and stable to treatment with hydroxylamine. Following trypsin digestion of labeled enzyme, two labeled peptides corresponding to 45% of the adduct were isolated and sequenced. The results demonstrated the presence of a single modified amino acid. Tandem mass spectrometry suggested that the modified amino acid is tyrosine, which is linked to the inhibitor in an unprecedented fashion.

A non-heme iron protein with heme tendencies: an investigation of the substrate specificity of thymine hydroxylase.

Thymine hydroxylase from Rhodotorula glutinis catalyzes the oxidation of thymine to its alcohol, aldehyde, and carboxylic acid in three successive reactions. Each step involves stoichiometric consumption of O2 and alpha-ketoglutarate and formation of CO2 and succinate. Given the promiscuity of this enzyme, it was hoped that it would serve as a prototype for understanding the mechanism of this class of enzymes, the non-heme Fe2+ dioxygenases. Kinetic parameters for thymine, O2, Fe2+, and alpha-ketoglutarate have been determined, and isotope effect analysis of (trideuteriomethyl)thymine with enzyme reveals D(V) = 2.08 and D(V/K) = 1.11 at saturating O2. The kinetic parameters for (hydroxymethyl)uracil oxidation have been determined, and incubation of (5'-R)- and (5'-S)-[5'-2H]-5-(hydroxymethyl)uracil with enzyme reveals stereospecific removal of the pro-S hydrogen. No apparent isotope effect is observed in this reaction. The substrate specificity of this enzyme has been examined in detail. The enzyme can catalyze epoxidation, oxidation of a thioether to a sulfoxide and a sulfone, hydroxylation of an unactivated carbon-hydrogen bond, and oxidation of a methylamine to formaldehyde, as revealed through studies with 5-vinyluracil, 5-(methylthio)uracil, 5,6-dihydrothymine, and 1-methylthymine, respectively. In each case, the products were identified by gas chromatography-mass spectrometry, and 18O2-labeling studies revealed that one atom from O2 is incorporated into each product. The enzyme has also been shown to catalyze an uncoupling of hydroxylation and decarboxylation in the presence of a substrate analog incapable of undergoing hydroxylation or a substrate that is difficult to oxidize.(ABSTRACT TRUNCATED AT 250 WORDS)