Enzymatic methods may underestimate the total serum bile acid concentration.

Enzymatic assays based on bacterial 3α-hydroxysteroid dehydrogenase are the method of choice for quantification of total bile acids (BAs) in serum. Although non-specific, it is generally considered precise and robust. The aim of this study was to investigate how changes in the BA spectrum might affect the reliability of the method. We measured standard solutions of twenty-three human and murine BAs using a commercial enzymatic assay and compared the measured vs. expected concentrations. Additionally, total BA concentrations in rat and human cholestatic samples with an abnormal BA spectrum were measured using an enzymatic assay, and a more specific LC-MS/MS method. We observed a great variability in the response of individual BAs in the enzymatic assay. Relative signal intensities ranged from 100% in glycocholic acid (reference) to only 20% in α-muricholic acid. The enzymatic assay markedly underestimated the BA concentrations in both human and rat cholestatic sera when compared to the LC-MS/MS assay. Our study indicated that the performance of an enzymatic assay largely depends on the BA spectrum, and the total concentration of BAs can be markedly underestimated. Samples with an atypical BA spectrum (viz. in rodents) should preferably be measured by other methods.

Site-specific observation of the conformational change of a protein with 15N-labeled Tyr residues using NMR.

One of the reasons it is difficult to analyze protein structural dynamics at atomic resolution using NMR is the molecular size of the protein. The selective amino acid labeling method is one of the effective methods that can solve this problem. In this study, to determine the site-specific conformational change in 3α-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831 (Ps3αHSD), which forms a dimer composed of two 26 kDa subunits, we expressed and purified 15N-Tyr labeled Ps3αHSD and its mutants, and analyzed the conformational change upon NADH binding. Using the Tyr substituted mutants, we first assigned the respective signals of four Tyr residues. In the titration experiments with NADH, the four Tyr signals changed uniquely; changes in chemical shift and signal broadening were observed. The NADH binding affinity, determined from the plots of the 1H and 15N chemical shift changes, was comparable to those reported previously. Together with the crystal structure information for Ps3αHSD in the NADH-free and -bound states, site-specific conformational changes including environmental changes could be deduced.

Human 3α-hydroxysteroid dehydrogenase type 3: structural clues of 5α-DHT reverse binding and enzyme down-regulation decreasing MCF7 cell growth.

Human 3α-HSD3 (3α-hydroxysteroid dehydrogenase type 3) plays an essential role in the inactivation of the most potent androgen 5α-DHT (5α-dihydrotestosterone). The present study attempts to obtain the important structure of 3α-HSD3 in complex with 5α-DHT and to investigate the role of 3α-HSD3 in breast cancer cells. We report the crystal structure of human 3α-HSD3·NADP(+)·A-dione (5α-androstane-3,17-dione)/epi-ADT (epiandrosterone) complex, which was obtained by co-crystallization with 5α-DHT in the presence of NADP(+) Although 5α-DHT was introduced during the crystallization, oxidoreduction of 5α-DHT occurred. The locations of A-dione and epi-ADT were identified in the steroid-binding sites of two 3α-HSD3 molecules per crystal asymmetric unit. An overlay showed that A-dione and epi-ADT were oriented upside-down and flipped relative to each other, providing structural clues for 5α-DHT reverse binding in the enzyme with the generation of different products. Moreover, we report the crystal structure of the 3α-HSD3·NADP(+)·4-dione (4-androstene-3,17-dione) complex. When a specific siRNA (100 nM) was used to suppress 3α-HSD3 expression without interfering with 3α-HSD4, which shares a highly homologous active site, the 5α-DHT concentration increased, whereas MCF7 cell growth was suppressed. The present study provides structural clues for 5α-DHT reverse binding within 3α-HSD3, and demonstrates for the first time that down-regulation of 3α-HSD3 decreases MCF7 breast cancer cell growth.

Expression, purification and functional characterization of a novel 3α-hydroxysteroid dehydrogenase from Pseudomonas aeruginosa.

3α-Hydroxysteroid dehydrogenase (3α-HSD) catalyzes the oxidation of the 3-hydroxyl group of steroids. The enzymatic conversion is a critical step in the enzymatic assay of urinary sulfated bile acids (SBAs), which is a valuable diagnosis index of hepatobiliary diseases. However, the source of 3α-HSD for clinical applications is limited. In this study, an open reading frame (ORF) encoding a novel 3α-HSD was successfully cloned from Pseudomonas aeruginosa and expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified by immobilized metal ion affinity chromatography. Enzyme characterization studies revealed that the protein has 3α-HSD activity and the Km value for sodium cholate is 1.06 mmol L(-1). More than 60% relative enzyme activity was observed in a wide range of pH and temperature, with an optimum pH at 8.0 and an optimum temperature at 30°C. The enzyme's good thermostability under 40°C would be favorable in clinical applications. Ion interference experiments indicated that Zn(2+) was an activating cofactor which increased the enzyme activity 1.75-fold. With the favorable characteristics mentioned above, the new 3α-HSD is a promising enzyme for clinical applications. More importantly, the present work is the first report on a 3α-HSD from P. aeruginosa.

Enzyme biosensor for androsterone based on 3α-hydroxysteroid dehydrogenase immobilized onto a carbon nanotubes/ionic liquid/NAD+ composite electrode.

A 3α-hydrosteroid biosensor for androsterone determination has been prepared by immobilizing the enzyme 3α-hydroxysteroid dehydrogenase (3α-HSD) in a composite electrode platform constituted of a mixture of multi-walled carbon nanotubes (MWCNTs), octylpyridinium hexafluorophosphate (OPPF(6)) ionic liquid and NAD(+) cofactor. This configuration allowed the fast, sensitive and stable electrochemical detection of the NADH generated in the enzyme reaction. All the experimental variables involved in the preparation and performance of the enzyme biosensor were optimized. Amperometry in stirred solutions at +400 mV provided a linear calibration plot for androsterone in the 0.5-10 µM concentration range with a slope value more than 200-times higher than that previously reported. The detection limit achieved was 0.15 µM and a low value of the apparent Michaelis-Menten constant (K(app)(M)), 36.0 µM, similar to that reported for the enzyme in solution, was calculated. The 3α-HSD/MWCNTs/OPPF(6)/NAD(+) biosensor provided good results in the determination of androsterone in spiked human serum samples.

The Del1 deposition domain can immobilize 3alpha-hydroxysteroid dehydrogenase in the extracellular matrix without interfering with enzymatic activity.

Developing methods that result in targeting of therapeutic molecules in gene therapies to target tissues has importance, as targeting can increase efficacy and decrease off target-side-effects. Work from my laboratory previously showed that the extracellular matrix protein Del1 is organized in the extracellular matrix (ECM) via the Del1 deposition domain (DDD). In this work, a fusion protein with DDD was made to assay the ability to immobilize an enzyme without disrupting enzymatic function. A prostatic cancer-derived cell line LNCap that grows in an androgen-dependent manner was used with 3alpha-hydroxysteroid dehydrogenase (3 alphaHD), which catalyzes dihydrotestosterone (DHT). Plasmids encoding a 3alphaHD:DDD fusion were generated and transfected into cultured cells. The effects of 3alphaHD immobilized in the ECM by the DDD were evaluated by monitoring growth of LNCap cells and DHT concentrations. It was demonstrated that the DDD could immobilize an enzyme in the ECM without interfering with function.

Characterization of local geometry of protein surfaces with the visibility criterion.

Experimentally determined protein tertiary structures are rapidly accumulating in a database, partly due to the structural genomics projects. Included are proteins of unknown function, whose function has not been investigated by experiments and was not able to be predicted by conventional sequence-based search. Those uncharacterized protein structures highlight the urgent need of computational methods for annotating proteins from tertiary structures, which include function annotation methods through characterizing protein local surfaces. Toward structure-based protein annotation, we have developed VisGrid algorithm that uses the visibility criterion to characterize local geometric features of protein surfaces. Unlike existing methods, which only concerns identifying pockets that could be potential ligand-binding sites in proteins, VisGrid is also aimed to identify large protrusions, hollows, and flat regions, which can characterize geometric features of a protein structure. The visibility used in VisGrid is defined as the fraction of visible directions from a target position on a protein surface. A pocket or a hollow is recognized as a cluster of positions with a small visibility. A large protrusion in a protein structure is recognized as a pocket in the negative image of the structure. VisGrid correctly identified 95.0% of ligand-binding sites as one of the three largest pockets in 5616 benchmark proteins. To examine how natural flexibility of proteins affects pocket identification, VisGrid was tested on distorted structures by molecular dynamics simulation. Sensitivity decreased approximately 20% for structures of a root mean square deviation of 2.0 A to the original crystal structure, but specificity was not much affected. Because of its intuitiveness and simplicity, the visibility criterion will lay the foundation for characterization and function annotation of local shape of proteins.

Elucidation of a complete kinetic mechanism for a mammalian hydroxysteroid dehydrogenase (HSD) and identification of all enzyme forms on the reaction coordinate: the example of rat liver 3alpha-HSD (AKR1C9).

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 3alpha-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.

Rat NAD+-dependent 3alpha-hydroxysteroid dehydrogenase (AKR1C17): a member of the aldo-keto reductase family highly expressed in kidney cytosol.

Mammalian 3alpha-hydroxysteroid dehydrogenases (3alpha-HSDs) have been divided into two types: Cytosolic NADP(H)-dependent 3alpha-HSDs belonging to the aldo-keto reductase family, and mitochondrial and microsomal NAD(+)-dependent 3alpha-HSDs belonging to the short-chain dehydrogenase/reductase family. In this study, we characterized a rat aldo-keto reductase (AKR1C17), whose functions are unknown. The recombinant AKR1C17 efficiently oxidized 3alpha-hydroxysteroids and bile acids using NAD(+) as the preferred coenzyme at an optimal pH of 7.4-9.5, and was inhibited by ketamine and organic anions. The mRNA for AKR1C17 was detected specifically in rat kidney, where the enzyme was more highly expressed as a cytosolic protein than NADP(H)-dependent 3alpha-HSD (AKR1C9). Thus, AKR1C17 represents a novel NAD(+)-dependent type of cytosolic 3alpha-HSD with unique inhibitor sensitivity and tissue distribution. In addition, the replacement of Gln270 and Glu276 of AKR1C17 with the corresponding residues of NADP(H)-dependent 3alpha-HSD resulted in a switch in favor of NADP(+) specificity, suggesting their key roles in coenzyme specificity.

Understanding oligomerization in 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni: an in silico approach and evidence for an active protein.

3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) from Comamonas testosteroni belongs to the short chain dehydrogenase/reductase (SDR) protein superfamily and catalyzes the oxidoreduction of a variety of steroid substrates, including the steroid antibiotic fusidic acid. The enzyme also mediates the carbonyl reduction of non-steroidal aldehydes and ketones such as a novel insecticide. It is suggested that 3alpha-HSD/CR contributes to the bioremediation of natural and synthetic toxicants by C. testosteroni. Crystallization and structure analysis showed that 3alpha-HSD/CR is active as a dimer. Dimerization takes place via an interface axis which has exclusively been observed in homotetrameric SDRs but never in the structure of a homodimeric SDR. The formation of a tetramer is blocked in 3alpha-HSD/CR by the presence of a predominantly alpha-helical subdomain which is missing in all other SDRs of known structure. For example, 3alpha/20beta-HSD from Streptomyces hydrogenans exhibits two main subunit interfaces arranged about two non-crystallographic two-fold axes which are perpendicular to each other and referred to as P and Q. This mode of dimerization is, however, sterically impossible in 3alpha-HSD/CR because of a 28 amino acids insertion into the classical Rossmann-fold motif between strand betaE and helix alphaF. This insertion is masking helices alphaE and alphaF, thus preventing the formation of a four helix bundle and enables the dimerization via a P-axis interface. This type of dimerization in SDRs has never been observed in a crystal structure so far. The aim of this study was to investigate whether the lack of this predominantly alpha-helical subdomain keeps 3alpha-HSD/CR to be an active enzyme and whether, by an in silico approach, the formation of a homotetramer or even a novel oligomerization mode can be expected. Redesign of this interface was performed on the basis of site directed mutagenesis and according to other SDR structures by an approach combining "in silico" and "wet chemistry". Simulations of sterical and structural effects after different mutations, by applying a combination of homology modelling and molecular dynamic simulations, provided an effective tool for extensive mutagenesis studies and indicated the possibility of tetramer formation of truncated 3alpha-HSD/CR. In addition, despite lacking the extra loop domain, mutant 3alpha-HSD/CR was shown to be active towards a variety of standard substrates.

Enhanced activity of 3alpha-hydroxysteroid dehydrogenase by addition of the co-solvent 1-butyl-3-methylimidazolium (L)-lactate in aqueous phase of biphasic systems for reductive production of steroids.

The enzyme activity of 3alpha-hydrosteroid dehydrogenase (HSDH) was enhanced by the addition of the co-solvent 1-butyl-3-methylimidazolium (L)-lactate ([Bmim][lactate]) to 50 mM Tris-HCl buffer. When utilizing [Bmim][lactate], the reaction velocity of HSDH increased. Also, reductive production of androsterone was investigated in an aqueous-organic solvent biphasic system containing 5% [Bmim][lactate] as the co-solvent of aqueous phase. In a coupled-enzyme system comprising HSDH and formate dehydrogenase (FDH), a two-fold increase in production rate of androsterone was obtained when utilizing [Bmim][lactate] with NADH regeneration.

Apo- and holo-structures of 3alpha-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831. Loop-helix transition induced by coenzyme binding.

Bacterial 3alpha-hydroxysteroid dehydrogenase, which belongs to a short-chain dehydrogenase/reductase family and forms a dimer composed of two 26-kDa subunits, catalyzes the oxidoreduction of hydroxysteroids in a coenzyme-dependent manner. This enzyme also catalyzes the oxidoreduction of nonsteroid compounds that play an important role in xenobiotic metabolism of bacteria. We performed an x-ray analysis on the crystal of Ps3alphaHSD, the enzyme from Pseudomonas sp. B-0831 complexed with NADH. The resulting crystal structure at 1.8A resolution showed that Ps3alphaHSD exists as a structural heterodimer composed of apo- and holo-subunits. A distinct structural difference between them was found in the 185-207-amino acid region, where the structure in the apo-subunit is disordered whereas that in the holo-subunit consists of two alpha-helices. This fact proved that the NADH binding allows the helical structures to form the substrate binding pocket even in the absence of the substrate, although the region corresponds to the so-called "substrate-binding loop." The induction of alpha-helices in solution by the coenzyme binding was also confirmed by the CD experiment. In addition, the CD experiment revealed that the helix-inducing ability of NADH is stronger than that of NAD. We discuss the negative cooperativity for the coenzyme binding, which is caused by the effect of the structural change transferred between the subunits of the heterodimer.

Chiral inversion of RS-8359: a selective and reversible MAO-A inhibitor via oxido-reduction of keto-alcohol.

RS-8359, (+/-)-4-(4-cyanoanilino)-5,6-dihydro-7-hydroxy-7H-cyclopenta[d]-pyrimidine is a selective and reversible MAO-A inhibitor. The (S)-enantiomer of RS-8359 has been demonstrated to be inverted to the (R)-enantiomer after oral administration to rats. In the current study, we investigated the chiral inversion mechanism and the properties of involved enzymes using rat liver subcellular fractions. The 7-hydroxy function of RS-8359 was oxidized at least by the two different enzymes. The cytosolic enzyme oxidized enantiospecifically the (S)-enantiomer with NADP as a cofactor. On the other hand, the microsomal enzyme catalyzed more preferentially the oxidation of the (S)-enantiomer than the (R)-enantiomer with NAD as a cofactor. With to product enantioselectivity of reduction of the 7-keto derivative, it was found that only the alcohol bearing (R)-configuration was formed by the cytosolic enzyme with NADPH and the microsomal enzyme with NADH at almost equal rate. The reduction rate was much larger than the oxidation rate of 7-hydroxy group. The results suggest that the chiral inversion might occur via an enantioselectivity of consecutive two opposing reactions, oxidation and reduction of keto-alcohol group. In this case, the direction of chiral inversion from the (S)-enantiomer to the (R)-enantiomer is governed by the enantiospecific reduction of intermediate 7-keto group to the alcohol with (R)-configuration. The enzyme responsible for the enantiospecific reduction of the 7-keto group was purified from rat liver cytosolic fractions and identified as 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) via database search of peptide mass data obtained by nano-LC/MS/MS.

Crystallization and preliminary X-ray analysis of the complex of NADH and 3alpha-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831.

The NAD(P)(+)-dependent enzyme 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) catalyzes the reversible interconversion of hydroxyl and oxo groups at position 3 of the steroid nucleus. The complex of NADH and 3alpha-HSD from Pseudomonas sp. B-0831 was crystallized by the hanging-drop vapour-diffusion method. Refinement of crystallization conditions with microseeding improved the quality of the X-ray diffraction data to a resolution of 1.8 A. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 62.46, b = 82.25, c = 86.57 A, and contained two molecules, reflecting dimer formation of 3alpha-HSD, in the asymmetric unit.

Molecular cloning of a novel type of rat cytoplasmic 17beta-hydroxysteroid dehydrogenase distinct from the type 5 isozyme.

Rat liver contains two cytosolic enzymes (TBER1 and TBER2) that reduce 6-tert-butyl-2,3-epoxy-5-cyclohexene-1,4-dione into its 4R- and 4S-hydroxy metabolites. In this study, we cloned the cDNA for TBER1 and examined endogenous substrates using the homogenous recombinant enzyme. The cDNA encoded a protein composed of 323 amino acids belonging to the aldo-keto reductase family. The recombinant TBER1 efficiently oxidized 17beta-hydroxysteroids and xenobiotic alicyclic alcohols using NAD+ as the preferred coenzyme at pH 7.4, and showed low activity towards 20alpha- and 3alpha-hydroxysteroids, and 9-hydroxyprostaglandins. The enzyme was potently inhibited by diethylstilbestrol, hexestrol and zearalenone. The coenzyme specificity, broad substrate specificity and inhibitor sensitivity of the enzyme differed from those of rat NADPH-dependent 17beta-hydroxysteroid dehydrogenase type 5, which was cloned from the liver and characterized using the recombinant enzyme. The mRNA for TBER1 was highly expressed in rat liver, gastrointestinal tract and ovary, in contrast to specific expression of 17beta-hydroxysteroid dehydrogenase type 5 mRNA in the liver and kidney. Thus, TBER1 represents a novel type of 17beta-hydroxysteroid dehydrogenase with unique catalytic properties and tissue distribution. In addition, TBER2 was identified as 3alpha-hydroxysteroid dehydrogenase on chromatographic analysis of the enzyme activities in rat liver cytosol and characterization of the recombinant 3alpha-hydroxysteroid dehydrogenase.

Purification and characterization of rat liver enzyme catalyzing stereoselective reduction of acetylpyridines.

Acetylpyridines (1-3) are known as aroma components of foods, perfumes, and smoking suppressants, showing several biological activities and constituting part of the structure of some important biologically active compounds. We purified and characterized an enzyme that catalyzes the stereoselective reduction of acetylpyridines so that we could clarify its function. The enzyme participating in the reductive metabolism of 4-acetylpyridine (1) in the rat liver was purified by successively applying ammonium sulfate fractionation, anion-exchange, gel filtration, and affinity chromatography, and it was definitively identified as 3alpha-HSD. It preferentially reduced acetylpyridines (1-3) and acetophenone (7) to their corresponding (S)-alcohols, with high enantioselectivity. Kinetic analyses of the compounds were performed, and the V(max)/K(m) values decreased in the order of 4-, 2-, and 3-acetylpyridine (1, 3, 2), while acetophenone (7) showed almost the same value as 3-acetylpyridine (2). These results suggested that the reduction of the substrates by 3alpha-HSD is affected by the nitrogen atom in the aromatic ring.

Comparison of crystal structures of human type 3 3alpha-hydroxysteroid dehydrogenase reveals an "induced-fit" mechanism and a conserved basic motif involved in the binding of androgen.

The aldo-keto reductase (AKR) human type 3 3alpha-hydroxysteroid dehydrogenase (h3alpha-HSD3, AKR1C2) plays a crucial role in the regulation of the intracellular concentrations of testosterone and 5alpha-dihydrotestosterone (5alpha-DHT), two steroids directly linked to the etiology and the progression of many prostate diseases and cancer. This enzyme also binds many structurally different molecules such as 4-hydroxynonenal, polycyclic aromatic hydrocarbons, and indanone. To understand the mechanism underlying the plasticity of its substrate-binding site, we solved the binary complex structure of h3alpha-HSD3-NADP(H) at 1.9 A resolution. During the refinement process, we found acetate and citrate molecules deeply engulfed in the steroid-binding cavity. Superimposition of this structure with the h3alpha-HSD3-NADP(H)-testosterone/acetate ternary complex structure reveals that one of the mobile loops forming the binding cavity operates a slight contraction movement against the citrate molecule while the side chains of many residues undergo numerous conformational changes, probably to create an optimal binding site for the citrate. These structural changes, which altogether cause a reduction of the substrate-binding cavity volume (from 776 A(3) in the presence of testosterone/acetate to 704 A(3) in the acetate/citrate complex), are reminiscent of the "induced-fit" mechanism previously proposed for the aldose reductase, another member of the AKR superfamily. We also found that the replacement of residues Arg(301) and Arg(304), localized near the steroid-binding cavity, significantly affects the 3alpha-HSD activity of this enzyme toward 5alpha-DHT and completely abolishes its 17beta-HSD activity on 4-dione. All these results have thus been used to reevaluate the binding mode of this enzyme for androgens.

The structure of Apo R268A human aldose reductase: hinges and latches that control the kinetic mechanism.

Aldose reductase (AR) catalyzes the NADPH-dependent reduction of glucose and other sugars to their respective sugar alcohols. The NADP+/NADPH exchange is the rate-limiting step for this enzyme and contributes in varying degrees to the catalytic rates of other aldo-keto reductase superfamily enzymes. The mutation of Arg268 to alanine in human recombinant AR removes one of the ligands of the C2-phosphate of NADP+ and markedly reduces the interaction of the apoenzyme with the nucleotide. The crystal structure of human R268A apo-aldose reductase determined to a resolution of 2.1 A is described. The R268A mutant enzyme has similar kinetic parameters to the wild-type enzyme for aldehyde substrates, yet has greatly reduced affinity for the nucleotide substrate which greatly facilitates its crystallization in the apoenzyme form. The apo-structure shows that a high temperature factor loop (between residues 214 and 226) is displaced by as much as 17 A in a rigid body fashion about Gly213 and Ser226 in the absence of the nucleotide cofactor as compared to the wild-type holoenzyme structure. Several factors act to stabilize the NADPH-holding loop in either the 'open' or 'closed' conformations: (1) the presence and interactions of the nucleotide cofactor, (2) the residues surrounding the Gly213 and Ser226 hinges which form unique hydrogen bonds in the 'open' or 'closed' structure, and (3) the Trp219 "latch" residue which interacts with an arginine residue, Arg293, in the 'open' conformation or with a cysteine residue, Cys298, in the 'closed' conformation. Several mutations in and around the high temperature factor loop are examined to elucidate the role of the loop in the mechanism by which aldose reductase binds and releases its nucleotide substrate.

An enzymatic cycling method for the determination of serum total bile acids with recombinant 3alpha-hydroxysteroid dehydrogenase.

A highly sensitive enzymatic cycling method was developed for the serum total bile acids assay. We constructed a prokaryotic expression system to prepare the recombinant 3alpha-hydroxysteroid dehydrogenase in place of the natural enzyme and for the first time used it in the total bile acids assay. The production rate of thio-NADH correlated with the bile acids concentration and was measured by the change of absorbance at 405/660 nm. The enzymatic cycling method could detect 0.22 micromol/L total bile acids in serum. Within-run and between-run imprecisions were 1.2-3.7% and 2.3-4.8%, respectively. The calibration curve for total bile acids in serum was linear between 0.5 and 180 micromol/L. This method was free from interference by bilirubin, hemoglobin, ascorbate, and lactate dehydrogenase. In conclusion, serum total bile acids could be measured by the enzymatic cycling method with recombinant 3alpha-hydroxysteroid dehydrogenase as the tool enzyme.

Dissection of the physiological interconversion of 5alpha-DHT and 3alpha-diol by rat 3alpha-HSD via transient kinetics shows that the chemical step is rate-determining: effect of mutating cofactor and substrate-binding pocket residues on catalysis.

3Alpha-hydroxysteroid dehydrogenases (3alpha-HSDs) catalyze the interconversion between 5alpha-dihydrotestosterone (5alpha-DHT), the most potent androgen, and 3alpha-androstanediol (3alpha-diol), a weak androgen metabolite. To identify the rate-determining step in this physiologically important reaction, rat liver 3alpha-HSD (AKR1C9) was used as the protein model for the human homologues in fluorescence stopped-flow transient kinetic and kinetic isotope effect studies. Using single and multiple turnover experiments to monitor the NADPH-dependent reduction of 5alpha-DHT, it was found that k(lim) and k(max) values were identical to k(cat), indicating that chemistry is rate-limiting overall. Kinetic isotope effect measurements, which gave (D)k(cat) = 2.4 and (D)2(O)k(cat) = 3.0 at pL 6.0, suggest that the slow chemical transformation is significantly rate-limiting. When the NADP(+)-dependent oxidation of 3alpha-diol was monitored, single and multiple turnover experiments showed a k(lim) and burst kinetics consistent with product release as being rate-limiting overall. When NAD(+) was substituted for NADP(+), burst phase kinetics was eliminated, and k(max) was identical to k(cat). Thus with the physiologically relevant substrates 5alpha-DHT plus NADPH and 3alpha-diol plus NAD(+), the slowest event is chemistry. R276 forms a salt-linkage with the phosphate of 2'-AMP, and when it is mutated, tight binding of NAD(P)H is no longer observed [Ratnam, K., et al. (1999) Biochemistry 38, 7856-7864]. The R276M mutant also eliminated the burst phase kinetics observed for the NADP(+)-dependent oxidation of 3alpha-diol. The data with the R276M mutant confirms that the release of the NADPH product is the slow event; and in its absence, chemistry becomes rate-limiting. W227 is a critical hydrophobic residue at the steroid binding site, and when it is mutated to alanine, k(cat)/K(m) for oxidation is significantly depressed. Burst phase kinetics for the NADP(+)-dependent turnover of 3alpha-diol by W227A was also abolished. In the W227A mutant, the slow release of NADPH is no longer observed since the chemical transformation is now even slower. Thus, residues in the cofactor and steroid-binding site can alter the rate-determining step in the NADP(+)-dependent oxidation of 3alpha-diol to make chemistry rate-limiting overall.