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L C Pedersen

Publications and source records attributed to L C Pedersen.

At least 19 recordsLinked to original sources
PubMed2003

Heparan sulphate N-sulphotransferase activity: reaction mechanism and substrate recognition.

Human heparan sulphate N-deacetylase/N-sulphotransferase 1 sulphates the NH(3) (+) group of the glucosamine moiety of the heparan chain in heparan sulphate/heparin biosynthesis. An open cleft that runs perpendicular to the sulphate donor 3'-phosphoadenosine 5'-phosphosulphate may constitute the acceptor substrate-binding site of the sulphotransferase domain (hNST1) [Kakuta, Sueyoshi, Negishi and Pedersen (1999) J. Biol. Chem. 274, 10673-10676]. When a hexasaccharide model chain is docked into the active site, only a trisaccharide (-IdoA-GlcN-IdoA-) portion interacts directly with the cleft residues: Trp-713, His-716 and His-720 from alpha helix 6, and Phe-640, Glu-641, Glu-642, Gln-644 and Asn-647 from random coil (residues 640-647). Mutation of these residues either abolishes or greatly reduces hNST1 activity. Glu-642 may play the critical role of catalytic base in the sulphuryl group transfer reaction, as indicated by its hydrogen-bonding distance to the NH(3) (+) group of the glucosamine moiety in the model and by mutational data.

Animals↗
PubMed2001

Structure and function of sulfotransferases.

Sulfotransferases (STs) catalyze the transfer reaction of the sulfate group from the ubiquitous donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to an acceptor group of numerous substrates. This reaction, often referred to as sulfuryl transfer, sulfation, or sulfonation, is widely observed from bacteria to humans and plays a key role in various biological processes such as cell communication, growth and development, and defense. The cytosolic STs sulfate small molecules such as steroids, bioamines, and therapeutic drugs, while the Golgi-membrane counterparts sulfate large molecules including glucosaminylglycans and proteins. We have now solved the X-ray crystal structures of four cytosolic and one membrane ST. All five STs are globular proteins composed of a single alpha/beta domain with the characteristic five-stranded beta-sheet. The beta-sheet constitutes the core of the Paps-binding and catalytic sites. Structural analysis of the PAPS-, PAP-, substrate-, and/or orthovanadate (VO(3-)(4))-bound enzymes has also revealed the common molecular mechanism of the transfer reaction catalyzed by sulfotransferses. The X-ray crystal structures have opened a new era for the study of sulfotransferases.

Binding Sites↗
PubMed2001

The dimerization motif of cytosolic sulfotransferases.

Cytosolic sulfotransferases sulfate steroids such as estrogens and hydroxysteroids. The enzymes, including human estrogen sulfotransferase (hEST) and hydroxysteroid sulfotransferase (hHST), are generally homodimers in solution with mouse estrogen sulfotransferase (mEST) being one of few exceptions. To identify the amino acid residues responsible for the dimerization, eight residues on the surface of hEST were mutated to their counterparts in mEST and mutated hESTs were then analyzed by gel filtration chromatography. A single mutation of Val(269) to Glu was sufficient to convert hEST to a monomer and the corresponding mutation of Val(260) also altered hHST to a monomer. The hHST crystal structure revealed a short stretch of peptide with the side-chains from two hHST monomers forming a hydrophobic zipper-like structure enforced by ion pairs at both ends. This peptide consisted of 10 residues near the C-terminus that, including the critical Val residue, is conserved as KXXXTVXXXE in nearly all cytosolic sulfotransferases. When mEST underwent the double mutations Pro269Thr/Glu270Val dimerization resulted. Thus, the KXXXTVXXXE sequence appears to be the common protein-protein interaction motif that mediates the homo- as well as heterodimerization of cytosolic sulfotransferases.

Amino Acid Motifs↗
PubMed2001

Crystal structure-based studies of cytosolic sulfotransferase.

Sulfation is a widely observed biological reaction conserved from bacterium to human that plays a key role in various biological processes such as growth, development, and defense against adversities. Deficiencies due to the lack of the ubiquitous sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) are lethal in humans. A large group of enzymes called sulfotransferases catalyze the transfer reaction of sulfuryl group of PAPS to the acceptor group of numerous biochemical and xenochemical substrates. Four X-ray crystal structures of sulfotransferases have now been determined: cytosolic estrogen, hydroxysteroid, aryl sulfotransferases, and a sulfotransferase domain of the Golgi-membrane heparan sulfate N-deacetylase/N-sulfotransferase 1. These have revealed the conserved core structure of the PAPS binding site, a common reaction mechanism, and some information concerning the substrate specificity. These crystal structures introduce a new era of the study of the sulfotransferases.

Animals↗
PubMed2000

Heparan/chondroitin sulfate biosynthesis. Structure and mechanism of human glucuronyltransferase I.

Human beta1,3-glucuronyltransferase I (GlcAT-I) is a central enzyme in the initial steps of proteoglycan synthesis. GlcAT-I transfers a glucuronic acid moiety from the uridine diphosphate-glucuronic acid (UDP-GlcUA) to the common linkage region trisaccharide Gal beta 1-3Gal beta 1-4Xyl covalently bound to a Ser residue at the glycosaminylglycan attachment site of proteoglycans. We have now determined the crystal structure of GlcAT-1 at 2.3 A in the presence of the donor substrate product UDP, the catalytic Mn(2+) ion, and the acceptor substrate analog Gal beta 1-3Gal beta 1-4Xyl. The enzyme is a alpha/beta protein with two subdomains that constitute the donor and acceptor substrate binding site. The active site residues lie in a cleft extending across both subdomains in which the trisaccharide molecule is oriented perpendicular to the UDP. Residues Glu(227), Asp(252), and Glu(281) dictate the binding orientation of the terminal Gal-2 moiety. Residue Glu(281) is in position to function as a catalytic base by deprotonating the incoming 3-hydroxyl group of the acceptor. The conserved DXD motif (Asp(194), Asp(195), Asp(196)) has direct interaction with the ribose of the UDP molecule as well as with the Mn(2+) ion. The key residues involved in substrate binding and catalysis are conserved in the glucuronyltransferase family as well as other glycosyltransferases.

Amino Acid Sequence↗
PubMed2000

Crystal structure of SULT2A3, human hydroxysteroid sulfotransferase.

The crystal structure of SULT2A3 human hydroxysteroid sulfotransferase has been solved at 2.4 A resolution in the presence of 3'-phosphoadenosine 5'-phosphate (PAP). The overall structure is similar to those of SULT1 enzymes such as estrogen sulfotransferase and the PAP binding site is conserved, however, significant differences exist in the positions of loops Pro14-Ser20, Glu79-Ile82 and Tyr234-Gln244 in the substrate binding pocket. Moreover, protein interaction in the crystal structure has revealed a possible dimer-directed conformational alteration that may regulate the SULT activity.

Amino Acid Sequence↗
PubMed2000

Heparan sulfate biosynthesis: a theoretical study of the initial sulfation step by N-deacetylase/N-sulfotransferase.

Heparan sulfate N-deacetylase/N-sulfotransferase (NDST) catalyzes the deacetylation and sulfation of N-acetyl-D-glucosamine residues of heparan sulfate, a key step in its biosynthesis. Recent crystallographic and mutational studies have identified several potentially catalytic residues of the sulfotransferase domain of this enzyme (, J. Biol. Chem. 274:10673-10676). We have used the x-ray crystal structure of heparan sulfate N-sulfotransferase with 3'-phosphoadenosine 5'-phosphate to build a solution model with cofactor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and a model heparan sulfate ligand bound, and subsequently performed a 2-ns dynamics solution simulation. The simulation results confirm the importance of residues Glu(642), Lys(614), and Lys(833), with the possible involvement of Thr(617) and Thr(618), in binding PAPS. Additionally, Lys(676) is found in close proximity to the reaction site in our solvated structure. This study illustrates for the first time the possible involvement of water in the catalysis. Three water molecules were found in the binding site, where they are coordinated to PAPS, heparan sulfate, and the catalytic residues.

Amidohydrolases↗
PubMed2000

Structure-function modeling of the interactions of N-alkyl-N-hydroxyanilines with rat hepatic aryl sulfotransferase IV.

Although previous investigations have clearly shown that N-hydroxy arylamines and N-hydroxy heterocyclic amines are substrates for sulfotransferases, relatively little is known about which structural features of the N-hydroxy arylamines are important for sulfation to occur. The purpose of this investigation was to determine the extent to which secondary N-alkyl-N-hydroxy arylamines interact with aryl sulfotransferase (AST) IV (also known as tyrosine-ester sulfotransferase or ST1A1) and to evaluate these interactions using molecular modeling techniques. AST IV is a major cytosolic sulfotransferase in the rat, and it catalyzes the sulfation of various phenols, benzylic alcohols, arylhydroxamic acids, oximes, and primary N-hydroxy arylamines. In this study, three secondary N-hydroxy arylamines, N-hydroxy-N-methylaniline, N-ethyl-N-hydroxyaniline, and N-hydroxy-N-n-propylaniline, were found to be substrates for the purified rat hepatic AST IV. However, when the N-alkyl substituent was an n-butyl group (i.e., N-n-butyl-N-hydroxyaniline), the interaction with the enzyme changed from that of a substrate to competitive inhibition. This change in specificity was further explored through the construction and use of a model for AST IV based on mouse estrogen sulfotransferase, an enzyme whose crystal structure has been previously determined to high resolution. Molecular modeling techniques were used to dock each of the above N-hydroxy arylamines into the active site of the homology model of AST IV and determine optimum ligand geometries. The results of these experiments indicated that steric constraints on the orientation of binding of secondary N-alkyl-N-hydroxy arylamines at the active site of AST IV play a significant role in determining the nature of the interaction of the enzyme with these compounds.

Aminophenols↗
PubMed1999

Substrate gating confers steroid specificity to estrogen sulfotransferase.

Estrogen sulfotransferase (EST) exhibits a high substrate specificity and catalytic efficiency toward estrogens such as estradiol (E2) but insignificant ability to sulfate hydroxysteroids such as dehydroepiandrosterone (DHEA). To provide the structural basis for this estrogen specificity, we mutated amino acid residues that constitute the substrate-binding site of EST. Among these mutants, only Tyr-81 decreased E2 and increased DHEA sulfotransferase activities. Substitution for Tyr-81 by smaller hydrophobic residues increased K(m(E2)) for E2 activity, whereas the k(cat(E2)) remained relatively constant. The Y81L mutant exhibited the same DHEA activity as wild-type hydroxysteroid sulfotransferase, for which K(m(DHEA)) remained relatively constant, and k(cat(DHEA)) was markedly increased. The side chain of Tyr-81 is directed at the A-ring of the E2 molecule in the substrate-binding pocket of EST, constituting a steric gate with Phe-142 sandwiching E2 from the opposite side. The present mutagenesis study indicates that the 3beta-hydroxyl group of the DHEA molecule is excluded from the catalytic site of EST through steric hindrance of Tyr-81 with the C-19 methyl group of DHEA. Thus, this stricture-like gating caused by steric hindrance appears to be a structural principle for conferring estrogen specificity to EST.

Dehydroepiandrosterone↗
PubMed1999

Structure and function of HNK-1 sulfotransferase. Identification of donor and acceptor binding sites by site-directed mutagenesis.

HNK-1 glycan, sulfo-->3GlcAbeta1-->3Galbeta1-->4GlcNAc-->R, is uniquely enriched in neural cells and natural killer cells and is thought to play important roles in cell-cell interaction. HNK-1 glycan synthesis is dependent on HNK-1 sulfotransferase (HNK-1ST), and cDNAs encoding human and rat HNK-1ST have been recently cloned. HNK-1ST belongs to the sulfotransferase gene family, which shares two homologous sequences in their catalytic domains. In the present study, we have individually mutated amino acid residues in these conserved sequences and determined how such mutations affect the binding to the donor substrate, adenosine 3'-phosphate 5'-phosphosulfate, and an acceptor. Mutations of Lys(128), Arg(189), Asp(190), Pro(191), and Ser(197) to Ala all abolished the enzymatic activity. When Lys(128) and Asp(190) were conservatively mutated to Arg and Glu, respectively, however, the mutated enzymes still maintained residual activity, and both mutant enzymes still bound to adenosine 3',5'-diphosphate-agarose. K128R and D190E mutant enzymes, on the other hand, exhibited reduced affinity to the acceptor as demonstrated by kinetic studies. These findings, together with those on the crystal structure of estrogen sulfotransferase and heparan sulfate N-deacetylase/sulfotransferase, suggest that Lys(128) may be close to the 3-hydroxyl group of beta-glucuronic acid in a HNK-1 acceptor. In contrast, the effect by mutation at Asp(190) may be due to conformational change because this amino acid and Pro(191) reside in a transition of the secondary structure of the enzyme. These results indicate that conserved amino acid residues in HNK-1ST play roles in maintaining a functional conformation and are directly involved in binding to donor and acceptor substrates.

Amino Acid Sequence↗
PubMed1999

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.

Amidohydrolases↗
PubMed1999

3'-Phosphoadenosine 5'-phosphosulfate binding site of flavonol 3-sulfotransferase studied by affinity chromatography and 31P NMR.

The function of Lys-59, Arg-141, and Arg-277 in PAPS binding and catalysis of the flavonol 3-sulfotransferase was investigated. Affinity chromatography of conservative mutants with PAPS analogues allowed us to determine that Lys-59 interacts with the 5' portion of the nucleotide, while Arg-141 interacts with the 3' portion, confirming assignments deduced from the crystal structure of mouse estrogen sulfotransferase [Kakuta, Y., Pedersen, L. G., Carter, C. W. , Negishi, M., and Pedersen, L. C. (1997) Nat. Struct. Biol. 4, 904-908]. The affinity chromatography method could be used to characterize site-directed mutants for other types of enzymes that bind nucleoside 3',5'- or 2',5'-diphosphates. 31P NMR spectra of enzyme-PAP complexes were recorded for the wild-type enzyme and K59R and K59A mutants. The results of these experiments suggest that Lys-59 is involved in the determination of the proper orientation of the phosphosulfate group for catalysis.

Adenosine Diphosphate↗
PubMed1999

Identification of the calcium binding site and a novel ytterbium site in blood coagulation factor XIII by x-ray crystallography.

The presence or absence of calcium determines the activation, activity, oligomerization, and stability of blood coagulation factor XIII. To explore these observed effects, we have determined the x-ray crystal structure of recombinant factor XIII A2 in the presence of calcium, strontium, and ytterbium. The main calcium binding site within each monomer involves the main chain oxygen atom of Ala-457, and also the side chains from residues Asn-436, Asp-438, Glu-485, and Glu-490. Calcium and strontium bind in the same location, while ytterbium binds several angstroms removed. A novel ytterbium binding site is also found at the dimer two-fold axis, near residues Asp-270 and Glu-272, and this site may be related to the reported inhibition by lanthanide metals (Achyuthan, K. E., Mary, A., and Greenberg, C. S. (1989) Biochem. J. 257, 331-338). The overall structure of ion-bound factor XIII is very similar to the previously determined crystal structures of factor XIII zymogen, likely due to the constraints of this monoclinic crystal form. We have merged the three independent sets of water molecules in the structures to determine which water molecules are conserved and possibly structurally significant.

Binding Sites↗
PubMed1998

Structural determinants of the bifunctional corn Hageman factor inhibitor: x-ray crystal structure at 1.95 A resolution.

Corn Hageman factor inhibitor (CHFI) is a bifunctional 127 residue, 13.6 kDa protein isolated from corn seeds. It inhibits mammalian trypsin and Factor XIIa (Hageman Factor) of the contact pathway of coagulation as well as alpha-amylases from several insect species. Among the plasma proteinases, CHFI specifically inhibits Factor XIIa without affecting the activity of other coagulation proteinases. We have isolated CHFI from corn and determined the crystallographic structure at 1.95 A resolution. Additionally, we have solved the structure of the recombinant protein produced in Escherichia coli at 2.2 A resolution. The two proteins are essentially identical. The proteinase binding loop is in the canonical conformation for proteinase inhibitors. In an effort to understand alpha-amylase inhibition by members of the family of 25 cereal trypsin/alpha-amylase inhibitors, we have made three-dimensional models of several proteins in the family based on the CHFI coordinates and the coordinates determined for wheat alpha-amylase inhibitor 0.19 [Oda, Y., Matsunaga, T., Fukuyama, K., Miyazaki, T., and Morimoto, T. (1997) Biochemistry 36, 13503-13511]. From an analysis of the models and a structure-based sequence analysis, we propose a testable hypothesis for the regions of these proteins which bind alpha-amylase. In the course of the investigations, we have found that the cereal trypsin/alpha-amylase inhibitor family is evolutionarily related to the family of nonspecific lipid-transfer proteins of plants. This is a new addition to the group which now consists of the trypsin/alpha-amylase inhibitors, 2S seed storage albumins, and the lipid-transfer family. Apparently, the four-helix conformation has been a successful vehicle in plant evolution for providing protection from predators, food for the embryo, and lipid transfer.

Amino Acid Sequence↗
PubMed1998

The sulfuryl transfer mechanism. Crystal structure of a vanadate complex of estrogen sulfotransferase and mutational analysis.

Estrogen sulfotransferase (EST) catalyzes transfer of the 5'-sulfuryl group of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) to the 3alpha-phenol group of estrogenic steroids such as estradiol (E2). The recent crystal structure of EST-adenosine 3', 5'-diphosphate (PAP)- E2 complex has revealed that residues Lys48, Thr45, Thr51, Thr52, Lys106, His108, and Try240 are in position to play a catalytic role in the sulfuryl transfer reaction of EST (Kakuta Y., Pedersen, L. G., Carter, C. W., Negishi, M., and Pedersen, L. C. (1997) Nat. Struct. Biol. 4, 904-908). Mutation of Lys48, Lys106, or His108 nearly abolishes EST activity, indicating that they play a critical role in catalysis. A present 2.2-A resolution structure of EST-PAP-vanadate complex indicates that the vanadate molecule adopts a trigonal bipyramidal geometry with its equatorial oxygens coordinated to these three residues. The apical positions of the vanadate molecule are occupied by a terminal oxygen of the 5'-phosphate of PAP (2.1 A) and a possible water molecule (2. 3 A). This water molecule superimposes well to the 3alpha-phenol group of E2 in the crystal structure of the EST.PAP.E2 complex. These structures are characteristic of the transition state for an in-line sulfuryl transfer reaction from PAPS to E2. Moreover, residues Lys48, Lys106, and His108 are found to be coordinated with the vanadate molecule at the transition state of EST.

Adenosine Diphosphate↗
PubMed1998

A role of Lys614 in the sulfotransferase activity of human heparan sulfate N-deacetylase/N-sulfotransferase.

An active sulfotransferase (ST, residues 558-882) domain of the human heparan sulfate N-deacetylase/N-sulfotransferase (hHSNST) has been identified by aligning the amino acid sequence of hHSNST to that of mouse estrogen sulfotransferase (EST). The bacterially expressed ST domain transfers the 5'-sulfuryl group of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to only deacetylated heparin with an efficiency similar to that previously reported for the purified rat HSNST. Moreover, the K(m,PAPS) (2.1 microM) of the ST domain is also similar to that of the rat enzyme. Lys48 is a key residue in mEST catalysis. The residue corresponding to Lys48 is conserved in all known heparan sulfate sulfotransferases (Lys614 in the ST domain of hHSNST). Mutation of Lys614 to Ala abolishes N-sulfotransferase activity, indicating an important catalytic role of Lys614 in the ST domain. Crystals of the ST domain have been grown (orthorhombic space group P2(1)2(1)2) with diffraction to 2.5 A resolution.

Alanine↗
PubMed1998

Mouse steroid sulfotransferases: substrate specificity and preliminary X-ray crystallographic analysis.

Three mouse cytosolic sulfotransferases were expressed in Escherichia coli cells in order to study their substrate specificities toward natural as well as synthetic steroid hormones. The Km and Vmax values confirmed the high substrate specificity of estrogen and hydroxysteroid sulfotransferases toward estradiol and dehydroepiandrosterone, respectively. In sharp contrast, the synthetic estrogen diethylstilbestrol was metabolized efficiently by both enzymes to its disulfate ester. These sulfotransferases display highly stereospecific sulfotransferase activity for sulfating only the trans-isomer of diethylstilbestrol. Crystals suitable for high-resolution structure determination of estrogen sulfotransferase were grown with polyethylene glycol. The crystals belong to the orthorhombic space group P2(1)2(1)2, and diffracted to 2.5 A.

Animals↗