Mechanism of posttranslational regulation of phenol sulfotransferase: expression of two enzyme forms through redox modification and nucleotide binding

Sulfotransferase catalyzes sulfuryl group transfer between a nucleotide and a variety of nucleophiles that may be sugar, protein, xenobiotics, and other small molecules. Nucleotides may serve as cosubstrate, cofactor, inhibitor, or regulator in an enzyme catalyzed sulfuryl group transfer reaction. We are trying to understand how nucleotide regulates the activity of phenol sulfotransferase (PST) through the expression of two enzyme forms. The homogeneous rat recombinant PST was obtained from Escherichia coli, and the nucleotide copurified was examined. The nucleotide was completely removed from inactive PST in high salt and oxidative condition. Total enzyme activity was recovered following incubation in reductive environment. Many nucleotides are known to tightly bind to PST but only one nucleotide, 3'-phosphoadenosine 5'-phosphate (PAP), was identified to combine with PST by ion-pair RP-HPLC, UV-visible spectra, (31)P NMR, and ESI-MS and MS-MS spectrometry. In addition to the presence or absence of PAP, oxidation following reduction of PST was required to completely interconvert the two forms of PST. According to the experimental results, a mechanism for the formation of the two enzyme forms was proposed.     

A single mutation converts the nucleotide specificity of phenol sulfotransferase from PAP to AMP

Sulfotransferases (STs) catalyze all the known biological sulfonations, in which a sulfuryl group from a common sulfonate donor such as 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is transferred to a nucleophilic acceptor. In addition to PAPS, phenol sulfotransferase (PST), a member of the ST family, utilizes other nucleotides as substrates with much less catalytic efficiency [Lin, E. S., and Yang, Y. S. (2000) Biochem. Biophys. Res. Commun. 271, 818-822]. Six amino acid residues of PST have been chosen for mutagenesis studies on the basis of a model of PST and its sequence alignment with those of available cytosolic and membrane-anchored STs. Systematic analyses of the mutants reveal that Ser134 is important for the regulation of nucleotide specificity between 3'-phosphoadenosine 5'-phosphate (PAP) and adenosine 5'-monophosphate (AMP). Kinetic studies also indicate that Ser134 plays a key role in nucleotide binding (K(m)) but not in catalysis (kcat). Consequently, the catalytic efficiency (kcat/K(m)) of PST can be altered by 5 orders of magnitude with a mutation of Ser134. Moreover, the change in nucleotide specificity from PAP to AMP can be achieved by mutation of Ser134 to any of the following residues: Glu, Gln, Arg, and His. Roles of Lys44, Arg126, and Arg253, which interact directly with the 5'- and 3'-phosphate of PAP, were also investigated by mutagenesis and kinetic experiments. On the basis of these findings, we suggest that Ser134 is the key residue that enables PST to discriminate PAP from AMP.     

Observation of a hybrid random ping-pong mechanism of catalysis for NodST: a mass spectrometry approach

An efficient enzyme kinetics assay using electrospray ionization mass spectrometry (ESI-MS) was initially applied to the catalytic mechanism investigation of a carbohydrate sulfotransferase, NodST. Herein, the recombinant NodST was overexpressed with a His(6)-tag and purified via Ni-NTA metal-affinity chromatography. In this bisubstrate enzymatic system, an internal standard similar in structure and ionization efficiency to the product was chosen in the ESI-MS assay, and a single point normalization factor was determined and used to quantify the product concentration. The catalytic mechanism of NodST was rapidly determined by fitting the MS kinetic data into a nonlinear regression analysis program. The initial rate kinetics analysis and product inhibition study described support a hybrid double-displacement, two-site ping-pong mechanism of NodST with formation of a sulfated NodST intermediate. This covalent intermediate was further isolated and detected via trypsin digestion and Fourier transform ion cyclotron resonance mass spectrometry. To our knowledge, these are the first mechanistic data reported for the bacterial sulfotransferase, NodST, which demonstrated the power of mass spectrometry in elucidating the reaction pathway and catalytic mechanism of promising enzymatic systems.     

Fluorometric assay for alcohol sulfotransferase

A sensitive fluorometric assay was developed for alcohol sulfotransferase (AST). This was the first continuous fluorometric assay reported for AST. It used 3'-phosphoadenosine 5'-phosphosulfate regenerated from 3-phosphoadenosine 5'-phosphate by a recombinant phenol sulfotransferase (PST) using 4-methylumbelliferyl sulfate as the sulfuryl group donor. The recombinant PST did not use the alcohol substrate under the designed condition, and the sensitivity for AST activity was found to be comparable to that of radioactive assay as reported in the literature. The change of fluorescence intensity of 4-methylumbelliferone corresponded directly to the amount of active AST and was sensitive enough to measure nanogram or picomole amounts of the enzyme activity. This fluorometric assay was used to determine the activities of AST as purified form and in crude extracts of pig liver, rat liver, and Escherichia coli. Some properties of human dehydroepiandrosterone sulfotransferase were determined by this method and were found to be comparable to published data. Under similar assay conditions, the contaminated activities of arylsulfatase in crude extracts were also determined. This method not only is useful for the routine and detailed kinetic study of this important class of enzymes but also has the potential for the development of a high-throughput procedure using microplate reader.     

Two Phenol Sulfotransferase Species from One cDNA: Nature of the Differences

A phenol sulfotransferase from rat liver (EC 2.8.2.9), expressed inEscherichia colifrom a single cDNA, was purified as two separable but catalytically active proteins. The proteins appeared to be identical to each other and to the natural liver sulfotransferase by comparison of their amino acid constitution, amino-terminal end group, and interaction with a polyclonal antibody raised against the liver enzyme. Each of the recombinant forms, α and β, catalyzed the sulfuryl group transfer from 4-nitrophenylsulfate to an acceptor phenol, a reaction in which 3′-phospho-adenosine 5′-phosphate (PAP) is a necessary intermediate. Only form β, however, catalyzes the physiological transfer of a sulfuryl group from 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to the free phenol. Evidence is presented that sulfotransferase α, but not β, has 1 mol of PAP tightly bound per enzyme dimer. The ability to utilize PAPS as a sulfate donor could be altered: form α could be treated and purified as form β to acquire the ability to use PAPS, whereas form β was treated by extended incubation with PAP, lost its ability to use PAPS, and was purified as form α.     

Golgi-resident PAP-specific 3'-phosphatase-coupled sulfotransferase assays

Sulfotransferases are a large group of enzymes that transfer a sulfonate group from the donor substrate, 3'-phosphoadenosine-5'-phosphosulfate (PAPS)(1), to various acceptor substrates, generating 3'-phosphoadenosine-5'-phosphate (PAP) as a by-product. A universal phosphatase-coupled sulfotransferase assay is described here. In this method, Golgi-resident PAP-specific 3'-phosphatase (gPAPP) is used to couple to a sulfotransferase reaction by releasing the 3'-phosphate from PAP. The released phosphate is then detected using malachite green reagents. The enzyme kinetics of gPAPP have been determined, which allows calculation of the coupling rate, the ratio of product-to-signal conversion, of the coupled reaction. This assay is convenient, as it eliminates the need for radioisotope labeling and substrate-product separation, and is more accurate through removal of product inhibition and correction of the results with the coupling rate. This assay is also highly reproducible, as a linear correlation factor above 0.98 is routinely achievable. Using this method, we measured the Michaelis-Menten constants for recombinant human CHST10 and SULT1C4 with the substrates phenolphthalein glucuronic acid and α-naphthol, respectively. The activities obtained with the method were also validated by performing simultaneous radioisotope assays. Finally, the removal of PAP product inhibition by gPAPP was clearly demonstrated in radioisotope assays.     

Transition state of the sulfuryl transfer reaction of estrogen sulfotransferase

Kinetic isotope effects have been measured for the estrogen sulfotransferase-catalyzed sulfuryl (SO3) transfer from p-nitrophenyl sulfate to the 5'-phosphoryl group of 3'-phosphoadenosine 5'-phosphate. 18(V/K)nonbridge = 1.0016 +/- 0.0005, 18(V/K)bridge = 1.0280 +/- 0.0006, and 15(V/K) = 1.0014 +/- 0.0004. (15(V/K) refers to the nitro group in p-nitrophenyl sulfate). The kinetic isotope effects indicate substantial S O bond fission in the transition state, with partial charge neutralization of the leaving group. The small kinetic isotope effect in the nonbridging sulfuryl oxygen atoms suggests no significant change in bond orders of these atoms occurs, consistent with modest nucleophilic involvement. A comparison of the data for enzymatic and uncatalyzed sulfuryl transfer reactions suggests that both proceed through very similar transition states.     

A continuous assay for the spectrophotometric analysis of sulfotransferases using aryl sulfotransferase IV.

We have developed a continuous spectrophotometric coupled-enzyme assay for sulfotransferase activity. This assay is based on the regeneration of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) from the desulfated 3'-phosphoadenosine-5'-phosphate (PAP) by a recombinant aryl sulfotransferase using p-nitrophenyl sulfate as the sulfate donor and visible spectrophotometric indicator of enzyme turnover. Here recombinant rat aryl sulfotransferase IV (AST-IV) is expressed, resolved to the pure beta-form during purification, and utilized for the regeneration. The activity of betaAST-IV to catalyze the synthesis of PAPS from PAP and p-nitrophenyl sulfate is demonstrated via capillary zone electrophoresis, and the kinetics of this reverse-physiological reaction are calculated. betaAST-IV is then applied to the coupled enzyme system, where the steady-state activity of the commercially available Nod factor sulfotransferase is verified with an enzyme concentration study and substrate-specificity assays of N-chitoses. The potential applications of this assay include rapid kinetic determinations for carbohydrate and protein sulfotransferases, high-throughput screening of potential sulfotransferase substrates and inhibitors, and biomedical screening of blood samples and other tissues for specific sulfotransferase enzyme activity and substrate concentration.     

Structural analysis of the sulfotransferase (3-o-sulfotransferase isoform 3) involved in the biosynthesis of an entry receptor for herpes simplex virus 1

Heparan sulfate (HS) plays essential roles in assisting herpes simplex virus infection and other biological processes. The biosynthesis of HS includes numerous specialized sulfotransferases that generate a variety of sulfated saccharide sequences, conferring the selectivity of biological functions of HS. We report a structural study of human HS 3-O-sulfotransferase isoform 3 (3-OST-3), a key sulfotransferase that transfers a sulfuryl group to a specific glucosamine in HS generating an entry receptor for herpes simplex virus 1. We have obtained the crystal structure of 3-OST-3 at 1.95 A in a ternary complex with 3'-phosphoadenosine 5'-phosphate and a tetrasaccharide substrate. Mutational analyses were also performed on the residues involved in the binding of the substrate. Residues Gln255 and Lys368 are essential for the sulfotransferase activity and lie within hydrogen bonding distances to the carboxyl and sulfo groups of the uronic acid unit. These residues participate in the substrate recognition of 3-OST-3. This structure provides atomic level evidence for delineating the substrate recognition and catalytic mechanism for 3-OST-3.     

Crystal structure of the sulfotransferase domain of human heparan sulfate N-deacetylase/ N-sulfotransferase 1

Heparan sulfate N-deacetylase/N-sulfotransferase (HSNST) catalyzes the first and obligatory step in the biosynthesis of heparan sulfates and heparin. The crystal structure of the sulfotransferase domain (NST1) of human HSNST-1 has been determined at 2.3-A resolution in a binary complex with 3'-phosphoadenosine 5'-phosphate (PAP). NST1 is approximately spherical with an open cleft, and consists of a single alpha/beta fold with a central five-stranded parallel beta-sheet and a three-stranded anti-parallel beta-sheet bearing an interstrand disulfide bond. The structural regions alpha1, alpha6, beta1, beta7, 5'-phosphosulfate binding loop (between beta1 and alpha1), and a random coil (between beta8 and alpha13) constitute the PAP binding site of NST1. The alpha6 and random coil (between beta2 and alpha2), which form an open cleft near the 5'-phosphate of the PAP molecule, may provide interactions for substrate binding. The conserved residue Lys-614 is in position to form a hydrogen bond with the bridge oxygen of the 5'-phosphate.     

Reaction product affinity regulates activation of human sulfotransferase 1A1 PAP sulfation

Cytosolic sulfotransferase (SULT)-catalyzed sulfation regulates the activity of bio-signaling molecules and aids in metabolizing hydroxyl-containing xenobiotics. The sulfuryl donor for the SULT reaction is adenosine 3′-phosphate 5′-phosphosulfate (PAPS), while products are adenosine 3′,5′-diphosphate (PAP) and a sulfated alcohol. Human phenol sulfotransferase (SULT1A1) is one of the major detoxifying enzymes for phenolic xenobiotics. The mechanism of SULT1A1-catalyzed sulfation of PAP by pNPS was investigated. PAP was sulfated by para-nitrophenyl sulfate (pNPS) in a concentration-dependent manner. 2-Naphthol inhibited sulfation of PAP, competing with pNPS, while phenol activated the sulfation reaction. At saturating PAP, a ping pong kinetic mechanism is observed with pNPS and phenol as substrates, consistent with phenol intercepting the E–PAPS complex prior to dissociation of PAPS. At high concentrations, phenol competes with pNPS, consistent with formation of the E–PAP–phenol dead-end complex. Data are consistent with the previously reported mechanism for sulfation of 2-naphthol by PAPS, and its activation by pNPS [14]. Overall, data are consistent with release of PAP from E–PAP and PAPS from E–PAPS contributing to rate-limitation in both reaction directions.     

Kinetic measurements and mechanism determination of Stf0 sulfotransferase using mass spectrometry

Mycobacterial carbohydrate sulfotransferase Stf0 catalyzes the sulfuryl group transfer from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to trehalose. The sulfation of trehalose is required for the biosynthesis of sulfolipid-1, the most abundant sulfated metabolite found in Mycobacterium tuberculosis. In this paper, an efficient enzyme kinetics assay for Stf0 using electrospray ionization (ESI) mass spectrometry is presented. The kinetic constants of Stf0 were measured, and the catalytic mechanism of the sulfuryl group transfer reaction was investigated in initial rate kinetics and product inhibition experiments. In addition, Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry was employed to detect the noncovalent complexes, the Stf0-PAPS and Stf0-trehalose binary complexes, and a Stf0-3'-phosphoadenosine 5'-phosphate-trehalose ternary complex. The results from our study strongly suggest a rapid equilibrium random sequential Bi-Bi mechanism for Stf0 with formation of a ternary complex intermediate. In this mechanism, PAPS and trehalose bind and their products are released in random fashion. To our knowledge, this is the first detailed mechanistic data reported for Stf0, which further demonstrates the power of mass spectrometry in elucidating the reaction pathway and catalytic mechanism of promising enzymatic systems.     

A novel mammalian lithium-sensitive enzyme with a dual enzymatic activity, 3'-phosphoadenosine 5'-phosphate phosphatase and inositol-polyphosphate 1-phosphatase.

We report the molecular cloning in Rattus norvegicus of a novel mammalian enzyme (RnPIP), which shows both 3'-phosphoadenosine 5'-phosphate (PAP) phosphatase and inositol-polyphosphate 1-phosphatase activities. This enzyme is the first PAP phosphatase characterized at the molecular level in mammals, and it represents the first member of a novel family of dual specificity enzymes. The phosphatase activity is strictly dependent on Mg2+, and it is inhibited by Ca2+ and Li+ ions. Lithium chloride inhibits the hydrolysis of both PAP and inositol-1,4-bisphosphate at submillimolar concentration; therefore, it is possible that the inhibition of the human homologue of RnPIP by lithium ions is related to the pharmacological action of lithium. We propose that the PAP phosphatase activity of RnPIP is crucial for the function of enzymes sensitive to inhibition by PAP, such as sulfotransferase and RNA processing enzymes. Finally, an unexpected connection between PAP and inositol-1,4-bisphosphate metabolism emerges from this work.     

Purification and characterization of bile salt sulfotransferase from human liver

Bile salt sulfotransferase, the enzyme responsible for the formation of bile salt sulfate esters, was purified extensively from normal human liver. The purification procedure included DEAE-Sephadex chromatography, taurocholate-agarose affinity chromatography, and preparative isoelectrofocusing. The final preparation had a specific activity of 18 nmol min-1 mg protein-1, representing a 760-fold purification from the cytosol fraction with a overall yield of 15%. The human enzyme has a Mr of 67,000 and a pI of 5.2. DEAE-Sephadex chromatography of the cytosol fraction revealed only a single species of activity. The limiting Km for the sulfuryl donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS), is 0.7 microM. The limiting Km for the sulfuryl acceptor, glycolithocholate (GLC), is 2 microM. Reciprocal plots were intersecting. Product inhibition studies established that adenosine 3',5'-diphosphate (PAP) was competitive with PAPS (Ki = 0.2 microM) and noncompetitive with respect to GLC. GLC sulfate was competitive with GLC (Ki = 2.2 microM) and noncompetitive with respect to PAPS. Also, 3-ketolithocholate, a dead-end inhibitor, was competitive with GLC (Ki = 0.6 microM) and noncompetitive with respect to PAPS. Iso-PAP (the 2' isomer of PAP) was competitive with PAPS (Ki = 0.3 microM) and noncompetitive with GLC. The cumulative results of the steady-state kinetics experiments point to a random mechanism for the binding of substrates and release of products. The purified enzyme displays no activity toward estrone, testosterone, or phenol. Among the reactive substrates tested, the Vmax/Km values are in the order GLC greater than 3-beta OH-5-cholenic acid greater than glycochenodeoxycholate greater than glycocholate. p-Chloromercuribenzoate inactivated the enzyme. Either PAPS or GLC protected against inactivation, suggesting the presence of a sulfhydryl group at the active site.     

Rv2131c from Mycobacterium tuberculosis is a CysQ 3'-phosphoadenosine-5'-phosphatase

Mycobacterium tuberculosis ( Mtb) produces a number of sulfur-containing metabolites that contribute to its pathogenesis and ability to survive in the host. These metabolites are products of the sulfate assimilation pathway. CysQ, a 3'-phosphoadenosine-5'-phosphatase, is considered an important regulator of this pathway in plants, yeast, and other bacteria. By controlling the pools of 3'-phosphoadenosine 5'-phosphate (PAP) and 3'-phosphoadenosine 5'-phosphosulfate (PAPS), CysQ has the potential to modulate flux in the biosynthesis of essential sulfur-containing metabolites. Bioinformatic analysis of the Mtb genome suggests the presence of a CysQ homologue encoded by the gene Rv2131c. However, a recent biochemical study assigned the protein's function as a class IV fructose-1,6-bisphosphatase. In the present study, we expressed Rv2131c heterologously and found that the protein dephosphorylates PAP in a magnesium-dependent manner, with optimal activity observed between pH 8.5 and pH 9.5 using 0.5 mM MgCl 2. A sensitive electrospray ionization mass spectrometry-based assay was used to extract the kinetic parameters for PAP, revealing a K m (8.1 +/- 3.1 microM) and k cat (5.4 +/- 1.1 s (-1)) comparable to those reported for other CysQ enzymes. The second-order rate constant for PAP was determined to be over 3 orders of magnitude greater than those determined for myo-inositol 1-phosphate (IMP) and fructose 1,6-bisphosphate (FBP), previously considered to be the primary substrates of this enzyme. Moreover, the ability of the Rv2131c-encoded enzyme to dephosphorylate PAP and PAPS in vivo was confirmed by functional complementation of an Escherichia coli Delta cysQ mutant. Taken together, these studies indicate that Rv2131c encodes a CysQ enzyme that may play a role in mycobacterial sulfur metabolism.     

X-ray crystal structure of human dopamine sulfotransferase, SULT1A3. Molecular modeling and quantitative structure-activity relationship analysis demonstrate a molecular basis for sulfotransferase substrate specificity

Humans are one of the few species that produce large amounts of catecholamine sulfates, and they have evolved a specific sulfotransferase, SULT1A3 (M-PST), to catalyze the formation of these conjugates. An orthologous protein has yet to be found in other species. To further our understanding of the molecular basis for the unique substrate selectivity of this enzyme, we have solved the crystal structure of human SULT1A3, complexed with 3'-phosphoadenosine 5'-phosphate (PAP), at 2.5 A resolution and carried out quantitative structure-activity relationship (QSAR) analysis with a series of phenols and catechols. SULT1A3 adopts a similar fold to mouse estrogen sulfotransferase, with a central five-stranded beta-sheet surrounded by alpha-helices. SULT1A3 is a dimer in solution but crystallized with a monomer in the asymmetric unit of the cell, although dimer interfaces were formed by interaction across crystallographic 2-fold axes. QSAR analysis revealed that the enzyme is highly selective for catechols, and catecholamines in particular, and that hydrogen bonding groups and lipophilicity (cLogD) strongly influenced K(m). We also investigated further the role of Glu(146) in SULT1A3 using site-directed mutagenesis and showed that it plays a key role not only in defining selectivity for dopamine but also in preventing many phenolic xenobiotics from binding to the enzyme.     

Crystal structure of the human estrogen sulfotransferase-PAPS complex: evidence for catalytic role of Ser137 in the sulfuryl transfer reaction

Estrogen sulfotransferase (EST) transfers the sulfate group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to estrogenic steroids. Here we report the crystal structure of human EST (hEST) in the context of the V269E mutant-PAPS complex, which is the first structure containing the active sulfate donor for any sulfotransferase. Superimposing this structure with the crystal structure of hEST in complex with the donor product 3'-phosphoadenosine 5'-phosphate (PAP) and the acceptor substrate 17beta-estradiol, the ternary structure with the PAPS and estradiol molecule, is modeled. These structures have now provided a more complete view of the S(N)2-like in-line displacement reaction catalyzed by sulfotransferases. In the PAPS-bound structure, the side chain nitrogen of the catalytic Lys(47) interacts with the side chain hydroxyl of Ser(137) and not with the bridging oxygen between the 5'-phosphate and sulfate groups of the PAPS molecule as is seen in the PAP-bound structures. This conformational change of the side chain nitrogen indicates that the interaction of Lys(47) with Ser(137) may regulate PAPS hydrolysis in the absences of an acceptor substrate. Supporting the structural data, the mutations of Ser(137) to cysteine and alanine decrease gradually k(cat) for PAPS hydrolysis and transfer activity. Thus, Ser(137) appears to play an important role in regulating the side chain interaction of Lys(47) with the bridging oxygen between the 5'-phosphate and the sulfate of PAPS.     

Inhibition of bovine phenol sulfotransferase (bSULT1A1) by CoA thioesters. Evidence for positive cooperativity and inhibition by interaction with both the nucleotide and phenol binding sites

Previous work with the bovine phenol sulfotransferase (bSULT1A1, EC ) demonstrated inhibition by CoA that was competitive with respect to the sulfuryl donor substrate, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) (Leach, M., Cameron, E., Fite, N., Stassinopoulos, J., Palmreuter, N., and Beckmann, J. D. (1999) Biochem. Biophys. Res. Commun. 261, 815-819). Here we report that long chain acyl-CoAs are more potent inhibitors of bSULT1A1 and also of human dopamine sulfotransferase (SULT1A3) when compared with unesterified CoA and short chain-length acyl-CoAs. A complex pattern of inhibition was revealed by systematic variation of palmitoyl-CoA, PAPS, and 7-hydroxycoumarin, the acceptor substrate. Convex plots of apparent K(m)/V(max) versus [palmitoyl-CoA] were adequately modeled using an ordered rapid equilibrium scheme with PAPS as the leading substrate and by accounting for the possible binding of two equivalents of inhibitor to the dimeric enzyme. Interestingly, the first K(i) of 2-3 microm was followed by a second K(i) of only 0.01-0.05 microm, suggesting that positive subunit cooperativity enhances binding of long chain acyl-CoAs to this sulfotransferase. Simultaneous interaction of palmitoyl-CoA with both the nucleotide and phenol binding sites is suggested by two experiments. First, the acyl-CoA displaced 7-hydroxycoumarin from the highly fluorescent bSULT1A1.PAP.7-HC complex in a cooperative manner. Second, palmitoyl-CoA prevented the quenching of bSULT1A1 fluorescence observed with pentachlorophenol. Finally, titrations of bSULT1A1-pentachlorophenol complex with palmitoyl-CoA caused the return of protein fluorescence, and the binding of palmitoyl-CoA was highly cooperative (Hill constant of 1.9). Overall, these results suggest a model of sulfotransferase inhibition in which the 3'-phosphoadenosine-5'-diphosphate moiety of CoA docks to the PAPS domain, and the acyl-pantetheine group docks to the hydrophobic phenol binding domain.     

The structures of the unique sulfotransferase retinol dehydratase with product and inhibitors provide insight into enzyme mechanism and inhibition

The structure of retinol dehydratase (DHR) from Spodoptera frugiperda, a member of the sulfotransferase superfamily, in complexes with the inactive form of the cofactor PAP 3'-phosphoadenosine 5'-phosphate (PAP) and (1) the product of the reaction with retinol anhydroretinol (AR), (2) the retinoid inhibitor all-trans-4-oxoretinol (OR), and (3) the potent steroid inhibitor androsterone (AND) have been determined and compared to the enzyme complex with PAP and retinol. The structures show that the geometry of the active-site amino acids is largely preserved in the various complexes. However, the beta-ionone rings of the retinoids are oriented differently with respect to side chains that have been shown to be important for the enzymatic reaction. In addition, the DHR:PAP:AND complex reveals a novel mode for steroid binding that contrasts significantly with that for steroid binding in other sulfotransferases. The molecule is displaced and rotated approximately 180 degrees along its length so that there is no acceptor hydroxyl in close proximity to the site of sulfate transfer. This observation explains why steroids are potent inhibitors of retinol dehydratase activity, rather than substrates for sulfonation. Most of the steroid-protein contacts are provided by the alpha-helical cap that distinguishes this member of the superfamily. This observation suggests that in addition to providing a chemical environment that promotes the dehydration of a sulfonated intermediate, the cap may also serve to minimize a promiscuous sulfotransferases activity.     

A novel target of lithium therapy

Phosphatases converting 3'-phosphoadenosine 5'-phosphate (PAP) into adenosine 5'-phosphate are of fundamental importance in living cells as the accumulation of PAP is toxic to several cellular systems. These enzymes are lithium-sensitive and we have characterized a human PAP phosphatase as a potential target of lithium therapy. A cDNA encoding a human enzyme was identified by data base screening, expressed in Escherichia coli and the 33 kDa protein purified to homogeneity. The enzyme exhibits high affinity for PAP (K(m)<1 microM) and is sensitive to subtherapeutic concentrations of lithium (IC(50)=0.3 mM). The human enzyme also hydrolyzes inositol-1, 4-bisphosphate with high affinity (K(m)=0.4 microM), therefore it can be considered as a dual specificity enzyme with high affinity (microM range) for both PAP and inositol-1,4-bisphosphate. Hydrolysis of inositol-1,4-bisphosphate was also inhibited by lithium (IC(50)=0.6 mM). Thus, we present experimental evidence for a novel target of lithium therapy, which could explain some of the side effects of this therapy.