Electric Fields and Enzyme Catalysis.

What happens inside an enzyme's active site to allow slow and difficult chemical reactions to occur so rapidly? This question has occupied biochemists' attention for a long time. Computer models of increasing sophistication have predicted an important role for electrostatic interactions in enzymatic reactions, yet this hypothesis has proved vexingly difficult to test experimentally. Recent experiments utilizing the vibrational Stark effect make it possible to measure the electric field a substrate molecule experiences when bound inside its enzyme's active site. These experiments have provided compelling evidence supporting a major electrostatic contribution to enzymatic catalysis. Here, we review these results and develop a simple model for electrostatic catalysis that enables us to incorporate disparate concepts introduced by many investigators to describe how enzymes work into a more unified framework stressing the importance of electric fields at the active site.

Supreme glutathione-dependent ketosteroid isomerase in the yellow-fever transmitting mosquito Aedes aegypti.

The steroid hormone ecdysone is essential for the reproduction and survival of insects. The hormone is synthesized from dietary sterols such as cholesterol, yielding ecdysone in a series of consecutive enzymatic reactions. In the insect orders Lepidoptera and Diptera a glutathione transferase called Noppera-bo (Nobo) plays an essential, but biochemically uncharacterized, role in ecdysteroid biosynthesis. The Nobo enzyme is consequently a possible target in harmful dipterans, such as disease-carrying mosquitoes. Flavonoid compounds inhibit Nobo and have larvicidal effects in the yellow-fever transmitting mosquito Aedes aegypti, but the enzyme is functionally incompletely characterized. We here report that within a set of glutathione transferase substrates the double-bond isomerase activity with 5-androsten-3,17-dione stands out with an extraordinary specific activity of 4000 µmol min-1 mg-1. We suggest that the authentic function of Nobo is catalysis of a chemically analogous ketosteroid isomerization in ecdysone biosynthesis.

Structure, Mutagenesis, and QM:MM Modeling of 3-Ketosteroid Δ1-Dehydrogenase from Sterolibacterium denitrificans─The Role of a New Putative Membrane-Associated Domain and Proton-Relay System in Catalysis.

3-Ketosteroid Δ1-dehydrogenases (KstD) are important microbial flavin enzymes that initiate the metabolism of steroid ring A and find application in the synthesis of steroid drugs. We present a structure of the KstD from Sterolibacterium denitrificans (AcmB), which contains a previously uncharacterized putative membrane-associated domain and extended proton-relay system. The experimental and theoretical studies show that the steroid Δ1-dehydrogenation proceeds according to the Ping-Pong bi-bi kinetics and a two-step base-assisted elimination (E2cB) mechanism. The mechanism is validated by evaluating the experimental and theoretical kinetic isotope effect for deuterium-substituted substrates. The role of the active-site residues is quantitatively assessed by point mutations, experimental activity assays, and QM/MM MD modeling of the reductive half-reaction (RHR). The pre-steady-state kinetics also reveals that the low pH (6.5) optimum of AcmB is dictated by the oxidative half-reaction (OHR), while the RHR exhibits a slight optimum at the pH usual for the KstD family of 8.5. The modeling confirms the origin of the enantioselectivity of C2-H activation and substrate specificity for Δ4-3-ketosteroids. Finally, the cholest-4-en-3-one turns out to be the best substrate of AcmB in terms of ΔG of binding and predicted rate of dehydrogenation.

Production of 21-hydroxy-20-methyl-pregna-1,4-dien-3-one by modifying multiple genes in Mycolicibacterium.

C22 steroid drug intermediates are suitable for corticosteroids synthesis, and the production of C22 steroids is unsatisfactory due to the intricate steroid metabolism. Among the C22 steroids, 21-hydroxy-20-methyl-pregna-1,4-dien-3-one (1,4-HP) could be used for Δ1-steroid drug synthesis, such as prednisolone. Nevertheless, the production of 1,4-HP remains unsatisfactory. In this study, an ideal 1,4-HP producing strain was constructed. By the knockout of 3-ketosteroid-9-hydroxylase (KshA) genes and 17ß-hydroxysteroid dehydrogenase (Hsd4A) gene, the steroid nucleus degradation and the accumulation of C19 steroids in Mycolicibacterium neoaurum were blocked. The mutant strain could transform phytosterols into 1,4-HP as the main product and 21-hydroxy-20-methyl-pregna-4-ene-3-one as a by-product. Subsequently, the purity of 1,4-HP improved to 95.2% by the enhancement of 3-ketosteroid-Δ1-dehydrogenase (KSTD) activity, and the production of 1,4-HP was improved by overexpressing NADH oxidase (NOX) and catalase (KATE) genes. Consequently, the yield of 1,4-HP achieved 10.5 g/L. The molar yield and the purity of 1,4-HP were optimal so far, and the production of 1,4-HP provides a new intermediate for the pharmaceutical steroid industry. KEY POINTS: • A third 3-ketosteroid-9-hydroxylase was identified in Mycolicibacterium neoaurum. • An 1,4-HP producer was constructed by KshA and Hsd4A deficiency. • The production of 1,4-HP was improved by KSTD, NOX, and KATE overexpression.

Inborn errors of bile acid metabolism in Japan.

Bile acids are a category of steroids biosynthesized from cholesterol in the liver. Inborn errors of their metabolism are inherited in an autosomal recessive manner, resulting in enzyme deficiencies affecting the bile acid biosynthetic pathway. These defects in the pathway cause accumulation of unusual bile acids or bile alcohols. Unusual bile acids are highly cytotoxic, causing injury to the liver. These unusual bile acids damage hepatocytes, resulting in cholestatic liver injury beginning in infancy. Except for cerebrotendinous xanthomatosis and some secondary defects, various inborn errors of bile acid metabolism (IEBAM) have been reported from Japan, affecting eight patients including three with 3ß-hydroxy-Δ5 -C27 -steroid dehydrogenase/isomerase deficiency, three with Δ4 -3-oxosteroid 5ß-reductase deficiency, one with oxysterol 7α-hydroxylase deficiency, and one with bile acid-CoA: amino acid N-acyltransferase deficiency. Distinctive laboratory findings in patients with 3ß-hydroxy-Δ5 -C27 -steroid dehydrogenase/isomerase deficiency, Δ4 -3-oxosteroid 5ß-reductase deficiency, and oxysterol 7α-hydroxylase deficiency include normal serum γ-glutamyltransferase and total bile acids concentrations despite presence of cholestasis (elevated serum direct bilirubin) from infancy. Pediatricians and pediatric surgeons who suspect a case of IEBAM should obtain urinary and serum bile acid analyses using gas or liquid chromatography-mass spectrometry as well as genetic analyses. Available treatments include oral cholic acid, chenodeoxycholic acid, glycocholic acid, and ursodeoxycholic acid; fat-soluble vitamin supplementation; and liver transplantation. Early diagnosis and treatment can offer a good outcome.

A novel nuclear receptor subfamily enlightens the origin of heterodimerization.

BACKGROUND: Nuclear receptors are transcription factors of central importance in human biology and associated diseases. Much of the knowledge related to their major functions, such as ligand and DNA binding or dimerization, derives from functional studies undertaken in classical model animals. It has become evident, however, that a deeper understanding of these molecular functions requires uncovering how these characteristics originated and diversified during evolution, by looking at more species. In particular, the comprehension of how dimerization evolved from ancestral homodimers to a more sophisticated state of heterodimers has been missing, due to a too narrow phylogenetic sampling. Here, we experimentally and phylogenetically define the evolutionary trajectory of nuclear receptor dimerization by analyzing a novel NR7 subgroup, present in various metazoan groups, including cnidarians, annelids, mollusks, sea urchins, and amphioxus, but lost in vertebrates, arthropods, and nematodes. RESULTS: We focused on NR7 of the cephalochordate amphioxus B. lanceolatum. We present a complementary set of functional, structural, and evolutionary analyses that establish that NR7 lies at a pivotal point in the evolutionary trajectory from homodimerizing to heterodimerizing nuclear receptors. The crystal structure of the NR7 ligand-binding domain suggests that the isolated domain is not capable of dimerizing with the ubiquitous dimerization partner RXR. In contrast, the full-length NR7 dimerizes with RXR in a DNA-dependent manner and acts as a constitutively active receptor. The phylogenetic and sequence analyses position NR7 at a pivotal point, just between the basal class I nuclear receptors that form monomers or homodimers on DNA and the derived class II nuclear receptors that exhibit the classical DNA-independent RXR heterodimers. CONCLUSIONS: Our data suggest that NR7 represents the "missing link" in the transition between class I and class II nuclear receptors and that the DNA independency of heterodimer formation is a feature that was acquired during evolution. Our studies define a novel paradigm of nuclear receptor dimerization that evolved from DNA-dependent to DNA-independent requirements. This new concept emphasizes the importance of DNA in the dimerization of nuclear receptors, such as the glucocorticoid receptor and other members of this pharmacologically important oxosteroid receptor subfamily. Our studies further underline the importance of studying emerging model organisms for supporting cutting-edge research.

Bioconversion of Phytosterols to 9-Hydroxy-3-Oxo-4,17-Pregadiene-20-Carboxylic Acid Methyl Ester by Enoyl-CoA Deficiency and Modifying Multiple Genes in Mycolicibacterium neoaurum.

Steroid drug precursors, including C19 and C22 steroids, are crucial to steroid drug synthesis and development. However, C22 steroids are less developed due to the intricacy of the steroid metabolic pathway. In this study, a C22 steroid drug precursor, 9-hydroxy-3-oxo-4,17-pregadiene-20-carboxylic acid methyl ester (9-OH-PDCE), was successfully obtained from Mycolicibacterium neoaurum by 3-ketosteroid-Δ1-dehydrogenase and enoyl-CoA hydratase ChsH deficiency. The production of 9-OH-PDCE was improved by the overexpression of 17ß-hydroxysteroid dehydrogenase Hsd4A and acyl-CoA dehydrogenase ChsE1-ChsE2 to reduce the accumulation of by-products. The purity of 9-OH-PDCE in fermentation broth was improved from 71.7% to 89.7%. Hence, the molar yield of 9-OH-PDCE was improved from 66.7% to 86.7%, with a yield of 0.78 g/L. Furthermore, enoyl-CoA hydratase ChsH1-ChsH2 was identified to form an indispensable complex in Mycolicibacterium neoaurum DSM 44704. IMPORTANCE C22 steroids are valuable precursors for steroid drug synthesis, but the development of C22 steroids remains unsatisfactory. This study presented a strategy for the one-step bioconversion of phytosterols to a C22 steroid drug precursor, 9-hydroxy-3-oxo-4,17-pregadiene-20-carboxylic acid methyl ester (9-OH-PDCE), by 3-ketosteroid-Δ1-dehydrogenase and enoyl-CoA hydratase deficiency with overexpression of 17ß-hydroxysteroid dehydrogenase acyl-CoA dehydrogenase in Mycolicibacterium. The function of the enoyl-CoA hydratase ChsH in vivo was revealed. Construction of the novel C22 steroid drug precursor producer provided more potential for steroid drug synthesis, and the characterization of the function of ChsH and the transformation of steroids further revealed the steroid metabolic pathway.

Construction of automated high-throughput screening method for finding efficient 3-ketosteroid 1,2-dehydrogenating strains.

Dehydrogenation reaction at C1(2) positions is typical and representative of industrial production of steroid drugs. Anti-inflammatory activity can be doubled when the nucleus of the anti-inflammatory steroid hormone drug introduces double bonds at the C1(2) positions. Arthrobacter simplex is currently the most widely studied and used strain for C1(2) dehydrogenation. Therefore, breeding Arthrobacter simplex with high-efficiency dehydrogenation ability is of great significance. In order to obtain high-efficiency strains, the research proposed a new screening strategy based on image process technique: firstly, a color reaction between 2,4-dinitrophenylhydrazine (DNPH) and 9α-hydroxyandrost-4-ene-3,17-dione (9α-OH-AD) was established to characterize the dehydrogenation ability of the strain; secondly, the color data of strains mutated by atmospheric and room temperature plasma (ARTP) in the "color reaction" were automated and analyzed for dehydrogenation ability prediction using optimized support vector machine model. Result showed that the prediction accuracy reached as high as 96% in verification experiments. After a series of mutagenesis, including breaking the bottleneck of a single mutation in ARTP, the dominant strain ARLU-146 was finally obtained from 5168 strains. Its initial conversion rate was 0.8059 g/L/h, with a conversion of 94.41% at 24 h, compared to the original strain ASP which increased the transformation rate by more than 10%. By further process optimization, a high conversion (94.34% within 20 h) with high substrate (85 g/L cortisone acetate) was achieved. According to literature research, it is the highest conversion at this substrate concentration. KEY POINTS: • A high-throughput screening method was developed by using image processing and machine learning technique. • "Mutation bottleneck" of single ARTP mutagenesis was surpassed by complex mutagenesis. • A high substrate (85 g/L CA) and high transformation rate craft (94.34% within 20 h) were built.

One-Step Chemo-, Regio- and Stereoselective Reduction of Ketosteroids to Hydroxysteroids over Zr-Containing MOF-808 Metal-Organic Frameworks.

Zr-containing MOF-808 is a very promising heterogeneous catalyst for the selective reduction of ketosteroids to the corresponding hydroxysteroids through a Meerwein-Ponndorf-Verley (MPV) reaction. Interestingly, the process leads to the diastereoselective synthesis of elusive 17α-hydroxy derivatives in one step, whereas most chemical and biological transformations produce the 17ß-OH compounds, or they require several additional steps to convert 17ß-OH into 17α-OH by inverting the configuration of the 17 center. Moreover, MOF-808 is found to be stable and reusable; it is also chemoselective (only keto groups are reduced, even in the presence of other reducible groups such as C=C bonds) and regioselective (in 3,17-diketosteroids only the keto group in position 17 is reduced, while the 3-keto group remains almost intact). The kinetic rate constant and thermodynamic parameters of estrone reduction to estradiol have been obtained by a detailed temperature-dependent kinetic analysis. The results evidence a major contribution of the entropic term, thus suggesting that the diastereoselectivity of the process is controlled by the confinement of the reaction inside the MOF cavities, where the Zr4+ active sites are located.

Universal capability of 3-ketosteroid Δ1-dehydrogenases to catalyze Δ1-dehydrogenation of C17-substituted steroids.

BACKGROUND: 3-Ketosteroid Δ1-dehydrogenases (KSTDs) are the enzymes involved in microbial cholesterol degradation and modification of steroids. They catalyze dehydrogenation between C1 and C2 atoms in ring A of the polycyclic structure of 3-ketosteroids. KSTDs substrate spectrum is broad, even though most of them prefer steroids with small substituents at the C17 atom. The investigation of the KSTD's substrate specificity is hindered by the poor solubility of the hydrophobic steroids in aqueous solutions. In this paper, we used 2-hydroxpropyl-ß-cyclodextrin (HBC) as a solubilizing agent in a study of the KSTDs steady-state kinetics and demonstrated that substrate bioavailability has a pivotal impact on enzyme specificity. RESULTS: Molecular dynamics simulations on KSTD1 from Rhodococcus erythropolis indicated no difference in ΔGbind between the native substrate, androst-4-en-3,17-dione (AD; - 8.02 kcal/mol), and more complex steroids such as cholest-4-en-3-one (- 8.40 kcal/mol) or diosgenone (- 6.17 kcal/mol). No structural obstacle for binding of the extended substrates was also observed. Following this observation, our kinetic studies conducted in the presence of HBC confirmed KSTD1 activity towards both types of steroids. We have compared the substrate specificity of KSTD1 to the other enzyme known for its activity with cholest-4-en-3-one, KSTD from Sterolibacterium denitrificans (AcmB). The addition of solubilizing agent caused AcmB to exhibit a higher affinity to cholest-4-en-3-one (Ping-Pong bi bi KmA = 23.7 µM) than to AD (KmA = 529.2 µM), a supposedly native substrate of the enzyme. Moreover, we have isolated AcmB isoenzyme (AcmB2) and showed that conversion of AD and cholest-4-en-3-one proceeds at a similar rate. We demonstrated also that the apparent specificity constant of AcmB for cholest-4-en-3-one (kcat/KmA = 9.25∙106 M-1 s-1) is almost 20 times higher than measured for KSTD1 (kcat/KmA = 4.71∙105 M-1 s-1). CONCLUSIONS: We confirmed the existence of AcmB preference for a substrate with an undegraded isooctyl chain. However, we showed that KSTD1 which was reported to be inactive with such substrates can catalyze the reaction if the solubility problem is addressed.

A Preorganized Electric Field Leads to Minimal Geometrical Reorientation in the Catalytic Reaction of Ketosteroid Isomerase.

Electrostatic interactions play a pivotal role in enzymatic catalysis and are increasingly modeled explicitly in computational enzyme design; nevertheless, they are challenging to measure experimentally. Using vibrational Stark effect (VSE) spectroscopy, we have measured electric fields inside the active site of the enzyme ketosteroid isomerase (KSI). These studies have shown that these fields can be unusually large, but it has been unclear to what extent they specifically stabilize the transition state (TS) relative to a ground state (GS). In the following, we use crystallography and computational modeling to show that KSI's intrinsic electric field is nearly perfectly oriented to stabilize the geometry of its reaction's TS. Moreover, we find that this electric field adjusts the orientation of its substrate in the ground state so that the substrate needs to only undergo minimal structural changes upon activation to its TS. This work provides evidence that the active site electric field in KSI is preorganized to facilitate catalysis and provides a template for how electrostatic preorganization can be measured in enzymatic systems.

Experimental and Computational Studies Unraveling the Peculiarity of Enolizable Oxoesters in the Organocatalyzed Mannich-Type Addition to Cyclic N-Acyl Iminium Ions.

γ- and δ-Oxoesters are easily available starting materials that have been sparingly used in some organocatalyzed reactions proceeding with a high enantioselectivity. In our experimentation we found that the use of these compounds as the enolizable (nucleophilic) component in organocatalyzed Mannich-type reactions using in situ-generated cyclic N-acyl iminium ions gave low diastereoselectivity and low to moderate values of enantioselectivity. This significant drop of facial selectivity with respect to simple aliphatic aldehydes has been rationalized by means of density functional theory (DFT) calculations.

Structural Coupling Throughout the Active Site Hydrogen Bond Networks of Ketosteroid Isomerase and Photoactive Yellow Protein.

Hydrogen bonds are fundamental to biological systems and are regularly found in networks implicated in folding, molecular recognition, catalysis, and allostery. Given their ubiquity, we asked the fundamental questions of whether, and to what extent, hydrogen bonds within networks are structurally coupled. To address these questions, we turned to three protein systems, two variants of ketosteroid isomerase and one of photoactive yellow protein. We perturbed their hydrogen bond networks via a combination of site-directed mutagenesis and unnatural amino acid substitution, and we used 1H NMR and high-resolution X-ray crystallography to determine the effects of these perturbations on the lengths of the two oxyanion hole hydrogen bonds that are donated to negatively charged transition state analogs. Perturbations that lengthened or shortened one of the oxyanion hole hydrogen bonds had the opposite effect on the other. The oxyanion hole hydrogen bonds were also affected by distal hydrogen bonds in the network, with smaller perturbations for more remote hydrogen bonds. Across 19 measurements in three systems, the length change in one oxyanion hole hydrogen bond was propagated to the other, by a factor of -0.30 ± 0.03. This common effect suggests that hydrogen bond coupling is minimally influenced by the remaining protein scaffold. The observed coupling is reproduced by molecular mechanics and quantum mechanics/molecular mechanics (QM/MM) calculations for changes to a proximal oxyanion hole hydrogen bond. However, effects from distal hydrogen bonds are reproduced only by QM/MM, suggesting the importance of polarization in hydrogen bond coupling. These results deepen our understanding of hydrogen bonds and their networks, providing strong evidence for long-range coupling and for the extent of this coupling. We provide a broadly predictive quantitative relationship that can be applied to and can be further tested in new systems.

Engineering of 3-ketosteroid-∆1-dehydrogenase based site-directed saturation mutagenesis for efficient biotransformation of steroidal substrates.

BACKGROUND: Biosynthesis of steroidal drugs is of great benefit in pharmaceutical manufacturing as the process involves efficient enzymatic catalysis at ambient temperature and atmospheric pressure compared to chemical synthesis. 3-ketosteroid-∆1-dehydrogenase from Arthrobacter simplex (KsdD3) catalyzes 1,2-desaturation of steroidal substrates with FAD as a cofactor. RESULTS: Recombinant KsdD3 exhibited organic solvent tolerance. W117, F296, W299, et al., which were located in substrate-binding cavity, were predicted to form hydrophobic interaction with the substrate. Structure-based site-directed saturation mutagenesis of KsdD3 was performed with W299 mutants, which resulted in improved catalytic activities toward various steroidal substrates. W299A showed the highest increase in catalytic efficiency (kcat/Km) compared with the wild-type enzyme. Homology modelling revealed that the mutants enlarged the active site cavity and relieved the steric interference facilitating recognition of C17 hydroxyl/carbonyl steroidal substrates. Steered molecular dynamics simulations revealed that W299A/G decreased the potential energy barrier of association of substrates and dissociation of the corresponding products. The biotransformation of AD with enzymatic catalysis and resting cells harbouring KsdD3 WT/mutants revealed that W299A catalyzed the maximum ADD yields of 71 and 95% by enzymatic catalysis and resting cell conversion respectively, compared with the wild type (38 and 75%, respectively). CONCLUSIONS: The successful rational design of functional KsdD3 greatly advanced our understanding of KsdD family enzymes. Structure-based site-directed saturation mutagenesis and biochemical data were used to design KsdD3 mutants with a higher catalytic activity and broader selectivity.

An Ab Initio QM/MM Study of the Electrostatic Contribution to Catalysis in the Active Site of Ketosteroid Isomerase.

The electric field in the hydrogen-bond network of the active site of ketosteroid isomerase (KSI) has been experimentally measured using vibrational Stark effect (VSE) spectroscopy, and utilized to study the electrostatic contribution to catalysis. A large gap was found in the electric field between the computational simulation based on the Amber force field and the experimental measurement. In this work, quantum mechanical (QM) calculations of the electric field were performed using an ab initio QM/MM molecular dynamics (MD) simulation and electrostatically embedded generalized molecular fractionation with conjugate caps (EE-GMFCC) method. Our results demonstrate that the QM-derived electric field based on the snapshots from QM/MM MD simulation could give quantitative agreement with the experiment. The accurate calculation of the electric field inside the protein requires both the rigorous sampling of configurations, and a QM description of the electrostatic field. Based on the direct QM calculation of the electric field, we theoretically confirmed that there is a linear correlation relationship between the activation free energy and the electric field in the active site of wild-type KSI and its mutants (namely, D103N, Y16S, and D103L). Our study presents a computational protocol for the accurate simulation of the electric field in the active site of the protein, and provides a theoretical foundation that supports the link between electric fields and enzyme catalysis.

Kemp Eliminase Activity of Ketosteroid Isomerase.

Kemp eliminases represent the most successful class of computationally designed enzymes, with rate accelerations of up to 109-fold relative to the rate of the same reaction in aqueous solution. Nevertheless, several other systems such as micelles, catalytic antibodies, and cavitands are known to accelerate the Kemp elimination by several orders of magnitude. We found that the naturally occurring enzyme ketosteroid isomerase (KSI) also catalyzes the Kemp elimination. Surprisingly, mutations of D38, the residue that acts as a general base for its natural substrate, produced variants that catalyze the Kemp elimination up to 7000-fold better than wild-type KSI does, and some of these variants accelerate the Kemp elimination more than the computationally designed Kemp eliminases. Analysis of the D38N general base KSI variant suggests that a different active site carboxylate residue, D99, performs the proton abstraction. Docking simulations and analysis of inhibition by active site binders suggest that the Kemp elimination takes place in the active site of KSI and that KSI uses the same catalytic strategies of the computationally designed enzymes. In agreement with prior observations, our results strengthen the conclusion that significant rate accelerations of the Kemp elimination can be achieved with very few, nonspecific interactions with the substrate if a suitable catalytic base is present in a hydrophobic environment. Computational design can fulfill these requirements, and the design of more complex and precise environments represents the next level of challenges for protein design.

Role of conserved Met112 residue in the catalytic activity and stability of ketosteroid isomerase.

Ketosteroid isomerase (3-oxosteroid Δ(5)-Δ(4)-isomerase, KSI) from Pseudomonas putida catalyzes allylic rearrangement of the 5,6-double bond of Δ(5)-3-ketosteroid to 4,5-position by stereospecific intramolecular transfer of a proton. The active site of KSI is formed by several hydrophobic residues and three catalytic residues (Tyr14, Asp38, and Asp99). In this study, we investigated the role of a hydrophobic Met112 residue near the active site in the catalysis, steroid binding, and stability of KSI. Replacing Met112 with alanine (yields M112A) or leucine (M112L) decreased the kcat by 20- and 4-fold, respectively. Compared with the wild type (WT), M112A and M112L KSIs showed increased KD values for equilenin, an intermediate analogue; these changes suggest that loss of packing at position 112 might lead to unfavorable steroid binding, thereby resulting in decreased catalytic activity. Furthermore, M112A and M112L mutations reduced melting temperature (Tm) by 6.4°C and 2.5°C, respectively. These changes suggest that favorable packing in the core is important for the maintenance of stability in KSI. The M112K mutation decreased kcat by 2000-fold, compared with the WT. In M112K KSI structure, a new salt bridge was formed between Asp38 and Lys112. This bridge could change the electrostatic potential of Asp38, and thereby contribute to the decreased catalytic activity. The M112K mutation also decreased the stability by reducing Tm by 4.1°C. Our data suggest that the Met112 residue may contribute to the catalytic activity and stability of KSI by providing favorable hydrophobic environments and compact packing in the catalytic core.

Δ4-3-ketosteroids as a new class of substrates for the cytosolic sulfotransferases.

Cytosolic sulfotransferase (SULT)-mediated sulfation is generally known to involve the transfer of a sulfonate group from the active sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), to a hydroxyl group or an amino group of a substrate compound. We report here that human SULT2A1, in addition to being able to sulfate dehydroepiandrosterone (DHEA) and other hydroxysteroids, could also catalyze the sulfation of Δ4-3-ketosteroids, which carry no hydroxyl groups in their chemical structure. Among a panel of Δ4-3-ketosteroids tested as substrates, 4-androstene-3,17-dione and progesterone were found to be sulfated by SULT2A1. Mass spectrometry analysis and structural modeling supported a reaction mechanism which involves the isomerization of Δ4-3-ketosteroids from the keto form to an enol form, prior to being subjected to sulfation. Results derived from this study suggested a potential role of SULT2A1 as a Δ4-3-ketosteroid sulfotransferase in steroid metabolism.

Molecular interactions of dioxins and DLCs with the ketosteroid receptors: an in silico risk assessment approach.

Dioxins and dioxin-like compounds (DLCs) are the ones with poor water solubility and low volatility, resistant to physical, chemical and biological processes, persistent in the environment even under extreme conditions. Due to lipophilic nature, they get adhered to the fatty material and concentrate through biomagnification and bioaccumulation, thereby easily getting incorporated into food chains, paving the way to endocrine disruption via modulation of various human receptors. This in turn leads to certain adverse health effects. In the present study, a total of 100 dioxins and DLCs were taken and their binding pattern was assessed with the ketosteroid receptors, i.e. androgen (hAR), glucocorticoid (hGR), progesterone (hPR) and mineralocorticoid (hMR) in comparison to the corresponding natural steroids and a known endocrine disrupting xenobiotic, Bisphenol A (BPA). Most of the DLCs, particularly those bearing hydroxyl (-OH) group showed considerable affinities with ketosteroid receptors. On comparing D scores of all the dioxins and DLCs against all four receptors, compound 8-hydroxy-3,4-dichlorodibenzofuran(8-OH-DCDF) exhibited least D score of -9.549 kcal mol-1 against hAR. 3,8-Dihydroxy-2-chlorodibenzofuran(3,8-DiOH-CDF), 4'-hydroxy-2,3,4,5-tetrachlorobiphenyl (4'-OH-TCB) and 4-hydroxy-2,2',5'-trichlorobiphenyl(4-OH-TCB) also showed comparable molecular interactions with the ketosteroid receptors. These interactions mainly include H-bonding, π-π stacking, hydrophobic, polar and van der Waals' interactions. In contrast, BPA and some natural ligands tested in this study showed lower binding affinities with these receptors than certain DLCs reported herein, i.e. certain DLCs might be more toxic than the proven toxic agent, BPA. Such studies play a pivotal role in the risk assessment of exposure to dioxins and DLCs on human health.

Structural and functional characterization of a ketosteroid transcriptional regulator of Mycobacterium tuberculosis.

Catabolism of host cholesterol is critical to the virulence of Mycobacterium tuberculosis and is a potential target for novel therapeutics. KstR2, a TetR family repressor (TFR), regulates the expression of 15 genes encoding enzymes that catabolize the last half of the cholesterol molecule, represented by 3aα-H-4α(3'-propanoate)-7aß-methylhexahydro-1,5-indane-dione (HIP). Binding of KstR2 to its operator sequences is relieved upon binding of HIP-CoA. A 1.6-Å resolution crystal structure of the KstR2(Mtb)·HIP-CoA complex reveals that the KstR2(Mtb) dimer accommodates two molecules of HIP-CoA. Each ligand binds in an elongated cleft spanning the dimerization interface such that the HIP and CoA moieties interact with different KstR2(Mtb) protomers. In isothermal titration calorimetry studies, the dimer bound 2 eq of HIP-CoA with high affinity (K(d) = 80 ± 10 nm) but bound neither HIP nor CoASH. Substitution of Arg-162 or Trp-166, residues that interact, respectively, with the diphosphate and HIP moieties of HIP-CoA, dramatically decreased the affinity of KstR2(Mtb) for HIP-CoA but not for its operator sequence. The variant of R162M that decreased the affinity for HIP-CoA (ΔΔG = 13 kJ mol(-1)) is consistent with the loss of three hydrogen bonds as indicated in the structural data. A 24-bp operator sequence bound two dimers of KstR2. Structural comparisons with a ligand-free rhodococcal homologue and a DNA-bound homologue suggest that HIP-CoA induces conformational changes of the DNA-binding domains of the dimer that preclude their proper positioning in the major groove of DNA. The results provide insight into KstR2-mediated regulation of expression of steroid catabolic genes and the determinants of ligand binding in TFRs.