New Developments in Selenium Biochemistry: Selenocysteine Biosynthesis in Eukaryotes and Archaea

We used comparative genomics and experimental analyses to show that (1) eukaryotes and archaea, which possess the selenocysteine (Sec) protein insertion machinery contain an enzyme, O-phosphoseryl-transfer RNA (tRNA)^[Ser]Sec kinase (designated PSTK), which phosphorylates seryl-tRNA^[Ser]Sec to form O-phosphoseryl-tRNA^[Ser]Sec and (2) the Sec synthase (SecS) in mammals is a pyridoxal phosphate-containing protein previously described as the soluble liver antigen (SLA). SecS uses the product of PSTK, O-phosphoseryl-tRNA^[Ser]Sec, and selenophosphate as substrates to generate selenocysteyl-tRNA^[Ser]Sec. Sec could be synthesized on tRNA^[Ser]Sec from selenide, adenosine triphosphate (ATP), and serine using tRNA^[Ser]Sec, seryl-tRNA synthetase, PSTK, selenophosphate synthetase, and SecS. The enzyme that synthesizes monoselenophosphate is a previously identified selenoprotein, selenophosphate synthetase 2 (SPS2), whereas the previously identified mammalian selenophosphate synthetase 1 did not serve this function. Monoselenophosphate also served directly in the reaction replacing ATP, selenide, and SPS2, demonstrating that this compound was the active selenium donor. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that contain selenoproteins. X.-M. Xu and B. A. Carlson contributed equally to the studies described herein.     

Active bovine selenophosphate synthetase 2, not having selenocysteine

During the course of studying selenocysteine (Sec) synthesis mechanisms in mammals, we prepared selenophosphate synthetase (SPS) from bovine liver by 4-step chromatography. In the last step of chromatography of hydroxyapatite, we found a protein band of molecular mass 33 kDa on SDS-PAGE, consistent with the pattern of SPS activity that was indirectly manifested by [⁷⁵Se]Sec production activity; however, we could not detect significant Se content in this active fraction. We also found a clear band of 33 kDa by Western blotting with antibody against a common peptide (387-401) in SPS2. We detected selenophosphate as the product of this active enzyme in the reaction mixture, composed of ATP, [⁷⁵Se]H₂Se and SPS. Chemically synthesized selenophosphate plays a role in Sec synthesis, not the addition of this enzyme. These results support that the product of SPS2 is selenophosphate itself. During this investigation, the probable sequence of bovine SPS2 not having Sec was reported in the blast information and the molecular mass was near with the protein in this report. Thus, bovine active SPS2 of molecular mass 33 kDa does not contain Sec. K. Furumiya and K. Kanaya contributed equally to this work.     

Crystal Structures of Catalytic Intermediates of Human Selenophosphate Synthetase 1

Selenophosphate synthetase catalyzes the synthesis of the highly active selenium donor molecule selenophosphate, a key intermediate in selenium metabolism. We have determined the high-resolution crystal structure of human selenophosphate synthetase 1 (hSPS1). An unexpected reaction intermediate, with a tightly bound phosphate and ADP at the active site has been captured in the structure. An enzymatic assay revealed that hSPS1 possesses low ADP hydrolysis activity in the presence of phosphate. Our structural and enzymatic results suggest that consuming the second high-energy phosphoester bond of ATP could protect the labile product selenophosphate during catalytic reaction. We solved another hSPS1 structure with potassium ions at the active sites. Comparing the two structures, we were able to define the monovalent cation-binding site of the enzyme. The detailed mechanism of the ADP hydrolysis step and the exact function of the monovalent cation for hSPS1 catalytic reaction are proposed.     

Binding of ATP and its derivatives to selenophosphate synthetase from <Emphasis Type="Italic">Escherichia coli</Emphasis>

Mechanistically similar selenophosphate synthetases (SPS) have been isolated from different organisms. SPS from Escherichia coli is an ATP-dependent enzyme with a C-terminal glycine-rich Walker sequence that has been assumed to take part in the first step of ATP binding. Three C-terminally truncated mutants of SPS, containing the N-terminal 238 (SPS₂₃₈), 262 (SPS₂₆₂), and 332 (SPS₃₃₂) amino acids of the 348-amino-acid protein, have been extracted from cell pellets, and two of these (SPS₂₆₂ and SPS₃₃₂) have been purified to homogeneity. SPS₂₃₈ has been obtained in a highly purified form. Binding of the fluorescent ATP-derivative TNP-ATP and Mn-ATP to the proteins was examined for all truncated mutants of SPS and a catalytically inactive C17S mutant. It has been shown that TNP-ATP can be used as a structural probe for ATP-binding sites of SPS. We observed two TNP-ATP binding sites per molecule of enzyme for wild-type SPS and SPS₃₃₂ mutant and one TNP-ATP binding site for SPS₂₃₈ mutant. The stoichiometry of Mn-ATP-binding was 2 mol of ATP per mol of protein determined with [¹⁴C]ATP by HPLC gel-filtration column chromatography under saturating conditions. The binding stoichiometries for SPS₃₃₂, SPS₂₆₂, and SPS₂₃₈ were 2, 1.6, and 1, respectively. The C17S mutant exhibits about one third of wild type SPS TNP-ATP-binding ability and converts 12% of ATP in the ATPase reaction to ADP in the absence of selenide. The C-terminus contributes two thirds to the TNP-ATP binding; SPS₂₃₈ likely has one ATP-binding site removed by truncation. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 8, pp. 1117–1125.     

Importance of Na,K-ATPase residue alpha 1-Arg544 in the segment Arg544-Asp567 for high-affinity binding of ATP, ADP, or MgATP

To identify residues involved in ATP binding in the N-domain of the alpha1-subunit of Na,K-ATPase, mutations were directed to the segment Arg(544)-Asp(567), a beta-strand-loop-helix structure with Arg(544) positioned at the mouth of the ATP-binding pocket near the interface to the P-domain. Substitution of Arg(544) with Gln abolished high-affinity binding of free ATP, while substitution with lysine reduced ADP affinity with minor effects on ATP binding. The contribution of Arg(544) to the change in free energy of ATP binding was estimated to 6.9 kJ/mol (DeltaDeltaG(b)) from double mutations with Asp(369) and to 7.8 kJ/mol from the MgATP dependence of phosphorylation. The phosphorylation data show that binding of Mg(2+) may increase the apparent affinity of wild-type enzyme for ATP [K(1/2)(ATP) 12 nM]. Moderately reduced affinities for ATP were seen after mutations of Asp(555), Glu(556), Asp(565), or Asp(567) with DeltaDeltaG(b) approximately equals 0.5-3 kJ/mol. Mutations of Cys(549) did not affect ATP binding. In conclusion, Arg(544) is important for binding of ATP or ADP, probably by stabilizing the beta- or gamma-phosphate moieties and aligning the gamma-phosphate for interaction with the carboxylate group of Asp(369).     

Structural insights into the catalytic mechanism of Escherichia coli selenophosphate synthetase

Selenophosphate synthetase (SPS) catalyzes the synthesis of selenophosphate, the selenium donor for the biosynthesis of selenocysteine and 2-selenouridine residues in seleno-tRNA. Selenocysteine, known as the 21st amino acid, is then incorporated into proteins during translation to form selenoproteins which serve a variety of cellular processes. SPS activity is dependent on both Mg(2+) and K(+) and uses ATP, selenide, and water to catalyze the formation of AMP, orthophosphate, and selenophosphate. In this reaction, the gamma phosphate of ATP is transferred to the selenide to form selenophosphate, while ADP is hydrolyzed to form orthophosphate and AMP. Most of what is known about the function of SPS has derived from studies investigating Escherichia coli SPS (EcSPS) as a model system. Here we report the crystal structure of the C17S mutant of SPS from E. coli (EcSPS(C17S)) in apo form (without ATP bound). EcSPS(C17S) crystallizes as a homodimer, which was further characterized by analytical ultracentrifugation experiments. The glycine-rich N-terminal region (residues 1 through 47) was found in the open conformation and was mostly ordered in both structures, with a magnesium cofactor bound at the active site of each monomer involving conserved aspartate residues. Mutating these conserved residues (D51, D68, D91, and D227) along with N87, also found at the active site, to alanine completely abolished AMP production in our activity assays, highlighting their essential role for catalysis in EcSPS. Based on the structural and biochemical analysis of EcSPS reported here and using information obtained from similar studies done with SPS orthologs from Aquifex aeolicus and humans, we propose a catalytic mechanism for EcSPS-mediated selenophosphate synthesis.     

Elucidation of the active conformation of the APS-kinase domain of human PAPS synthetase 1

Bifunctional human PAPS synthetase (PAPSS) catalyzes, in a two-step process, the formation of the activated sulfate carrier 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The first reaction involves the formation of the 5'-adenosine phosphosulfate (APS) intermediate from ATP and inorganic sulfate. APS is then further phosphorylated on its 3'-hydroxyl group by an additional ATP molecule to generate PAPS. The former reaction is catalyzed by the ATP-sulfurylase domain and the latter by the APS-kinase domain. Here, we report the structure of the APS-kinase domain of PAPSS isoform 1 (PAPSS1) representing the Michaelis complex with the products ADP-Mg and PAPS. This structure provides a rare glimpse of the active conformation of an enzyme catalyzing phosphoryl transfer without resorting to substrate analogs, inactivating mutations, or catalytically non-competent conditions. Our structure shows the interactions involved in the binding of the magnesium ion and PAPS, thereby revealing residues critical for catalysis. The essential magnesium ion is observed bridging the phosphate groups of the products. This function of the metal ion is made possible by the DGDN-loop changing its conformation from that previously reported, and identifies these loop residues unambiguously as a Walker B motif. Furthermore, the second aspartate residue of this motif is the likely candidate for initiating nucleophilic attack on the ATP gamma-phosphate group by abstracting the proton from the 3'-hydroxyl group of the substrate APS. We report the structure of the APS-kinase domain of human PAPSS1 in complex with two APS molecules, demonstrating the ability of the ATP/ADP-binding site to bind APS. Both structures reveal extended N termini that approach the active site of the neighboring monomer. Together, these results significantly increase our understandings of how catalysis is achieved by APS-kinase.     

Crystallographic studies on multiple conformational states of active-site loops in pyrrolysyl-tRNA synthetase

Pyrrolysine, a lysine derivative with a bulky pyrroline ring, is the “22nd” genetically encoded amino acid. In the present study, the carboxy-terminal catalytic fragment of Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS) was analyzed by X-ray crystallography and site-directed mutagenesis. The catalytic fragment ligated tRNA^Pyl with pyrrolysine nearly as efficiently as the full-length PylRS. We determined the crystal structures of the PylRS catalytic fragment in the substrate-free, ATP analogue (AMPPNP)-bound, and AMPPNP/pyrrolysine-bound forms, and compared them with the previously-reported PylRS structures. The ordering loop and the motif-2 loop undergo conformational changes from the “open” states to the “closed” states upon AMPPNP binding. On the other hand, the β7-β8 hairpin exhibits multiple conformational states, the open, intermediate (β7-open/β8-open and β7-closed/β8-open), and closed states, which are not induced upon substrate binding. The PylRS structures with a docked tRNA suggest that the active-site pocket can accommodate the CCA terminus of tRNA when the motif-2 loop is in the closed state and the β7-β8 hairpin is in the open or intermediate state. The entrance of the active-site pocket is nearly closed in the closed state of the β7-β8 hairpin, which may protect the pyrrolysyladenylate intermediate in the absence of tRNA^Pyl. Moreover, a structure-based mutational analysis revealed that hydrophobic residues in the amino acid-binding tunnel are important for accommodating the pyrrolysine side chain and that Asn346 is essential for anchoring the side-chain carbonyl and α-amino groups of pyrrolysine. In addition, a docking model of PylRS with tRNA was constructed based on the aspartyl-tRNA synthetase/tRNA structure, and was confirmed by a mutational analysis.     

Structural snapshots of the KMSKS loop rearrangement for amino acid activation by bacterial tyrosyl-tRNA synthetase

Tyrosyl-tRNA synthetase (TyrRS) has been studied extensively by mutational and structural analyses to elucidate its catalytic mechanism. TyrRS has the HIGH and KMSKS motifs that catalyze the amino acid activation with ATP. In the present study, the crystal structures of the Escherichia coli TyrRS catalytic domain, in complexes with l-tyrosine and a l-tyrosyladenylate analogue, Tyr-AMS, were solved at 2.0A and 2.7A resolution, respectively. In the Tyr-AMS-bound structure, the 2'-OH group and adenine ring of the Tyr-AMS are strictly recognized by hydrogen bonds. This manner of hydrogen-bond recognition is conserved among the class I synthetases. Moreover, a comparison between the two structures revealed that the KMSKS loop is rearranged in response to adenine moiety binding and hydrogen-bond formation, and the KMSKS loop adopts the more compact ("semi-open") form, rather than the flexible, open form. The HIGH motif initially recognizes the gamma-phosphate, and then the alpha and gamma-phosphates of ATP, with a slight rearrangement of the residues. The other residues around the substrate also accommodate the Tyr-AMS. This induced-fit form presents a novel "snapshot" of the amino acid activation step in the aminoacylation reaction by TyrRS. The present structures and the T.thermophilus TyrRS ATP-free and bound structures revealed that the extensive induced-fit conformational changes of the KMSKS loop and the local conformational changes within the substrate binding site form the basis for driving the amino acid activation step: the KMSKS loop adopts the open form, transiently shifts to the semi-open conformation according to the adenosyl moiety binding, and finally assumes the rigid ATP-bound, closed form. After the amino acid activation, the KMSKS loop adopts the semi-open form again to accept the CCA end of tRNA for the aminoacyl transfer reaction.     

Substrate induced structural and dynamics changes in human phosphomevalonate kinase and implications for mechanism

Phosphomevalonate kinase (PMK) catalyzes an essential step in the mevalonate pathway, which is the only pathway for synthesis of isoprenoids and steroids in humans. PMK catalyzes transfer of the gamma-phosphate of ATP to mevalonate 5-phosphate (M5P) to form mevalonate 5-diphosphate. Bringing these phosphate groups in proximity to react is especially challenging, given the high negative charge density on the four phosphate groups in the active site. As such, conformational and dynamics changes needed to form the Michaelis complex are of mechanistic interest. Herein, we report the characterization of substrate induced changes (Mg-ADP, M5P, and the ternary complex) in PMK using NMR-based dynamics and chemical shift perturbation measurements. Mg-ADP and M5P K(d)'s were 6-60 microM in all complexes, consistent with there being little binding synergy. Binding of M5P causes the PMK structure to compress (tau(c) = 13.5 nsec), whereas subsequent binding of Mg-ADP opens the structure up (tau(c) = 15.6 nsec). The overall complex seems to stay very rigid on the psec-nsec timescale with an average NMR order parameter of S(2) approximately 0.88. Data are consistent with addition of M5P causing movement around a hinge region to permit domain closure, which would bring the M5P domain close to ATP to permit catalysis. Dynamics data identify potential hinge residues as H55 and R93, based on their low order parameters and their location in extended regions that connect the M5P and ATP domains in the PMK homology model. Likewise, D163 may be a hinge residue for the lid region that is homologous to the adenylate kinase lid, covering the "Walker-A" catalytic loop. Binding of ATP or ADP appears to cause similar conformational changes; however, these observations do not indicate an obvious role for gamma-phosphate binding interactions. Indeed, the role of gamma-phosphate interactions may be more subtle than suggested by ATP/ADP comparisons, because the conservative O to NH substitution in the beta-gamma bridge of ATP causes a dramatic decrease in affinity and induces few chemical shift perturbations. In terms of positioning of catalytic residues, binding of M5P induces a rigidification of Gly21 (adjacent to the catalytically important Lys22), although exchange broadening in the ternary complex suggests some motion on a slower timescale does still occur. Finally, the first nine residues of the N-terminus are highly disordered, suggesting that they may be part of a cleavable signal or regulatory peptide sequence.     

Structure of anthrax edema factor-calmodulin-adenosine 5'-(alpha,beta-methylene)-triphosphate complex reveals an alternative mode of ATP binding to the catalytic site

Anthrax edema factor (EF) is a key virulence factor secreted by Bacillus anthracis. Here, we report a structure, at 3.0 A resolution, of the catalytic domain of EF (EF3) in complex with calmodulin (CaM) and adenosine 5'-(alpha,beta-methylene)-triphosphate (AMPCPP). Although the binding of the triphosphate of AMPCPP to EF3 can be superimposed on that of previously determined 3'deoxy-ATP (3'dATP) and 2'deoxy 3' anthraniloyl-ATP (2'd3' ANT-ATP) in EF3-CaM, the ribose and the adenine rings of AMPCPP are rotated approximately 105 and 180 degrees, respectively, relative to those of 3'dATP and 2'd3'ANT-ATP. Based on this model, K382 and F586 should play key roles in the recognition of adenine. However, mutations of these residues to alanine either separately or together cause only modest changes in Michaelis-Menten constants and IC50 values of AMPCPP and cAMP. Therefore, this alternate binding mode of the adenosine of AMPCPP binds to EF likely playing only a minor role in ATP binding and in catalysis.     

Hyperpolization-activated Ca channels in guard cell plasma membrane are involved in extracellular ATP-promoted stomatal opening in Vicia faba

Extracellular ATP (eATP) plays essential roles in plant growth, development, and stress tolerance. Extracellular ATP-regulated stomatal movement of Arabidopsis thaliana has been reported. Here, ATP was found to promote stomatal opening of Vicia faba in a dose-dependent manner. Three weakly hydrolysable ATP analogs (adenosine 5′-O-(3-thio) triphosphate (ATPγS), 3′-O-(4-benzoyl) benzoyl adenosine 5′-triphosphate (Bz-ATP) and 2-methylthio-adenosine 5′-triphosphate (2meATP)) showed similar effects, indicating that ATP acts as a signal molecule rather than an energy charger. ADP promoted stomatal opening, while AMP and adenosine did not affect stomatal movement. An ATP-promoted stomatal opening was blocked by the NADPH oxidase inhibitor diphenylene iodonium (DPI), the reductant dithiothreitol (DTT) or the Ca²⁺ channel blockers GdCl₃ and LaCl₃. A hyperpolarization-activated Ca²⁺ channel was detected in plasma membrane of guard cell protoplast. Extracellular ATP and weakly hydrolyzable ATP analogs activated this Ca²⁺ channel significantly. Extracellular ATP-promoted Ca²⁺ channel activation was markedly inhibited by DPI or DTT. These results indicated that eATP may promote stomatal opening via reactive oxygen species that regulate guard cell plasma membrane Ca²⁺ channels.     

ATP binding,not hydrolysis,at the first nucleotide-binding domain of multidrug resistance-associated protein MRP1 enhances ADP.Vi trapping at the second domain

Multidrug resistance-associated protein (MRP1) transports solutes in an ATP-dependent manner by utilizing its two nonequivalent nucleotide binding domains (NBDs) to bind and hydrolyze ATP. We found that ATP binding to the first NBD of MRP1 increases binding and trapping of ADP at the second domain (Hou, Y., Cui, L., Riordan, J. R., and Chang, X. (2002) J. Biol. Chem. 277, 5110-5119). These results were interpreted as indicating that the binding of ATP at NBD1 causes a conformational change in the molecule and increases the affinity for ATP at NBD2. However, we did not distinguish between the possibilities that the enhancement of ADP trapping might be caused by either ATP binding alone or hydrolysis. We now report the following. 1) ATP has a much lesser effect at 0 degrees C than at 37 degrees C. 2) After hexokinase treatment, the nonhydrolyzable ATP analogue, adenyl 5'-(yl iminodiphosphate), does not enhance ADP trapping. 3) Another nonhydrolyzable ATP analogue, adenosine 5'-(beta,gamma-methylene)triphosphate, whether hexokinase-treated or not, causes a slight enhancement. 4) In contrast, the hexokinase-treated poorly hydrolyzable ATP analogue, adenosine 5'-O-(thiotriphosphate) (ATPgammaS), enhances ADP trapping to a similar extent as ATP under conditions in which ATPgammaS should not be hydrolyzed. We conclude that: 1) ATP hydrolysis is not required to enhance ADP trapping by MRP1 protein; 2) with nucleotides having appropriate structure such as ATP or ATPgammaS, binding alone can enhance ADP trapping by MRP1; 3) the stimulatory effect on ADP trapping is greatly diminished when the MRP1 protein is in a "frozen state" (0 degrees C); and 4) the steric structure of the nucleotide gamma-phosphate is crucial in determining whether binding of the nucleotide to NBD1 of MRP1 protein can induce the conformational change that influences nucleotide trapping at NBD2.     

The roles of ADP2- and Mg2+ in control steps of phosphoglycerate kinase

1H-NMR measurements were made of solutions of yeast phosphoglycerate kinase containing the nucleotide, ADP, and Mg2+ in varying concentrations in order to investigate the affect that the metal ion has on the mode of ADP binding to the enzyme. A preliminary study of adenosine binding to phosphoglycerate kinase was made in order to be sure of the nature of the adenine site. From the change in chemical shifts of the 'basic patch' histidine resonances (His62, 167 and 170), the nucleotide C8-H, C2-H and C1'-H resonances and resonances 40 and 41 (assigned to Thr373 and Thr375 in the hydrophobic, i.e. catalytic, site), it is apparent that there are at least two ADP binding sites on the enzyme: one at the hydrophobic (catalytic) site and one at the electrostatic site. A comparison of the results for ADP and ATP reveals differences due to the differential binding of the phosphate groups. The presence of Mg2+ results in further differences being observed. The data suggest that the primary binding site of ADP, in the absence of Mg2+, involves electrostatic interactions between the diphosphate chain of the substrate and the 'basic patch' region of the N-terminal domain. In the presence of greater than or equal to 1:1 ratio of Mg2+/ADP, however, the primary binding site involves predominantly hydrophobic interactions between the adenosine moiety and the catalytic site, with secondary binding occurring at the electrostatic site. Addition of Mg2+, therefore, tends to reduce the affinity of the electrostatic site (presumably by competing for ADP). It is suggested that alpha-helix XII, including residues 372, 373 and 375, moves differentially on binding ADP, Mg ADP, ATP or Mg . ATP, consistent with Mg2+ assisting the transfer of the gamma-phosphate of ATP to 3-phosphoglycerate during catalysis.     

Exponential ATP amplification through simultaneous regeneration from AMP and pyrophosphate for luminescence detection of bacteria

Bacteria monitoring is essential for many industrial manufacturing processes, particularly those involving in food, biopharmaceuticals, and semiconductor production. Firefly luciferase ATP luminescence assay is a rapid and simple bacteria detection method. However, the detection limit of this assay for Escherichia coli is approximately 10⁴ colony-forming units (CFU), which is insufficient for many applications. This study aims to improve the assay sensitivity by simultaneous conversion of PP_i and AMP, two products of the luciferase reaction, back to ATP to form two chain-reaction loops. Because each consumed ATP continuously produces two new ATP molecules, this approach can achieve exponential amplification of ATP. Two consecutive enzyme reactions were employed to regenerate AMP into ATP: adenylate kinase converting AMP into ADP using UTP as the energy source, and acetate kinase catalyzing acetyl phosphate and ADP into ATP. The PP_i-recycling loop was completed using ATP sulfurylase and adenosine 5′ phosphosulfate. The modification maintains good quantification linearity in the ATP luminescence assay and greatly increases its bacteria detection sensitivity. This improved method can detect bacteria concentrations of fewer than 10 CFU. This exponential ATP amplification assay will benefit bacteria monitoring in public health and manufacturing processes that require high-quality water.     

Structure and catalytic mechanism of eukaryotic selenocysteine synthase

In eukaryotes and Archaea, selenocysteine synthase (SecS) converts O-phospho-L-seryl-tRNA [Ser]Sec into selenocysteyl-tRNA [Ser]Sec using selenophosphate as the selenium donor compound. The molecular mechanisms underlying SecS activity are presently unknown. We have delineated a 450-residue core of mouse SecS, which retained full selenocysteyl-tRNA [Ser]Sec synthesis activity, and determined its crystal structure at 1.65 A resolution. SecS exhibits three domains that place it in the fold type I family of pyridoxal phosphate (PLP)-dependent enzymes. Two SecS monomers interact intimately and together build up two identical active sites around PLP in a Schiff-base linkage with lysine 284. Two SecS dimers further associate to form a homotetramer. The N terminus, which mediates tetramer formation, and a large insertion that remodels the active site set SecS aside from other members of the family. The active site insertion contributes to PLP binding and positions a glutamate next to the PLP, where it could repel substrates with a free alpha-carboxyl group, suggesting why SecS does not act on free O-phospho-l-serine. Upon soaking crystals in phosphate buffer, a previously disordered loop within the active site insertion contracted to form a phosphate binding site. Residues that are strictly conserved in SecS orthologs but variant in related enzymes coordinate the phosphate and upon mutation corrupt SecS activity. Modeling suggested that the phosphate loop accommodates the gamma-phosphate moiety of O-phospho-l-seryl-tRNA [Ser]Sec and, after phosphate elimination, binds selenophosphate to initiate attack on the proposed aminoacrylyl-tRNA [Ser]Sec intermediate. Based on these results and on the activity profiles of mechanism-based inhibitors, we offer a detailed reaction mechanism for the enzyme.     

Biosynthesis of selenophosphate

Selenophosphate synthetase, the product of the selD gene, produces the highly active selenium donor, monoselenophosphate, from selenide and ATP. Positional isotope exchange experiments have shown hydrolysis of ATP occurs by way of a phosphoryl-enzyme intermediate. Although, mutagenesis studies have demonstrated Cys17 in the Escherichia coli enzyme is essential for catalytic activity the nucleophile in catalysis has not been identified. Recently, selenophosphate synthetase enzymes have been identified from other organisms. The human enzyme which contains a threonine residue corresponding to Cys17 in the E. coli enzyme, has been overexpressed in E. coli. The purified enzyme shows no detectable activity in the in vitro selenophosphate synthetase assay. In contrast, when the human enzyme is expressed to complement a selD mutation in E. coli, in the presence of 75Se, incorporation of 75Se into bacterial selenoproteins is observed. The inactive purified human enzyme together with the very low determined specific activity of the E. coli enzyme (83 nmol/min/mg) suggest an essential component for the formation of selenophosphate has not been identified.     

Activity of E. coli ClpA bound by nucleoside diphosphates and triphosphates

The Escherichia coli ClpA protein is a molecular chaperone that binds and translocates protein substrates into the proteolytic cavity of the tetradecameric serine protease ClpP. In the absence of ClpP, ClpA can remodel protein complexes. In order for ClpA to bind protein substrates targeted for removal or remodeling, ClpA requires nucleoside triphosphate binding to first assemble into a hexamer. Here we report the assembly properties of ClpA in the presence of the nucleoside diphosphates and triphosphates ADP, adenosine 5′-[γ-thio]triphosphate, adenosine 5′-(β,γ-imido)triphosphate, β,γ-methyleneadenosine 5′-triphosphate, and adenosine diphosphate beryllium fluoride. In addition to examining the assembly of ClpA in the presence of various nucleotides and nucleotide analogues, we have also correlated the assembly state of ClpA in the presence of these nucleotides with both polypeptide binding activity and enzymatic activity, specifically ClpA-catalyzed polypeptide translocation. Here we show that all of the selected nucleotides, including ADP, promote the assembly of ClpA. However, only adenosine 5′-[γ-thio]triphosphate and adenosine 5′-(β,γ-imido)triphosphate promote the formation of an oligomer of ClpA that is active in polypeptide binding and translocation. These results suggest that the presence of γ phosphate may serve to switch ClpA into a conformational state with high peptide binding activity, whereas affinity is severely attenuated when ADP is bound.     

Structural and kinetic analyses of arginine residues in the active site of the acetate kinase from Methanosarcina thermophila

Acetate kinase catalyzes transfer of the gamma-phosphate of ATP to acetate. The only crystal structure reported for acetate kinase is the homodimeric enzyme from Methanosarcina thermophila containing ADP and sulfate in the active site (Buss, K. A., Cooper, D. C., Ingram-Smith, C., Ferry, J. G., Sanders, D. A., and Hasson, M. S. (2001) J. Bacteriol. 193, 680-686). Here we report two new crystal structure of the M. thermophila enzyme in the presence of substrate and transition state analogs. The enzyme co-crystallized with the ATP analog adenosine 5'-[gamma-thio]triphosphate contained AMP adjacent to thiopyrophosphate in the active site cleft of monomer B. The enzyme co-crystallized with ADP, acetate, Al(3+), and F(-) contained a linear array of ADP-AlF(3)-acetate in the active site cleft of monomer B. Together, the structures clarify the substrate binding sites and support a direct in-line transfer mechanism in which AlF(3) mimics the meta-phosphate transition state. Monomers A of both structures contained ADP and sulfate, and the active site clefts were closed less than in monomers B, suggesting that domain movement contributes to catalysis. The finding that His(180) was in close proximity to AlF(3) is consistent with a role for stabilization of the meta-phosphate that is in agreement with a previous report indicating that this residue is essential for catalysis. Residue Arg(241) was also found adjacent to AlF(3), consistent with a role for stabilization of the transition state. Kinetic analyses of Arg(241) and Arg(91) replacement variants indicated that these residues are essential for catalysis and also indicated a role in binding acetate.     

Crystal structure of human phosphoribosylpyrophosphate synthetase 1 reveals a novel allosteric site

PRPP (phosphoribosylpyrophosphate) is an important metabolite essential for nucleotide synthesis and PRS (PRPP synthetase) catalyses synthesis of PRPP from R5P (ribose 5-phosphate) and ATP. The enzymatic activity of PRS is regulated by phosphate ions, divalent metal cations and ADP. In the present study we report the crystal structures of recombinant human PRS1 in complexes with SO4(2-) ions alone and with ATP, Cd2+ and SO4(2-) ions respectively. The AMP moiety of ATP binds at the ATP-binding site, and a Cd2+ ion binds at the active site and in a position to interact with the beta- and gamma-phosphates of ATP. A SO4(2-) ion, an analogue of the activator phosphate, was found to bind at both the R5P-binding site and the allosteric site defined previously. In addi-tion, an extra SO4(2-) binds at a site at the dimer interface between the ATP-binding site and the allosteric site. Binding of this SO4(2-) stabilizes the conformation of the flexible loop at the active site, leading to the formation of the active, open conformation which is essential for binding of ATP and initiation of the catalytic reaction. This is the first time that structural stabilization at the active site caused by binding of an activator has been observed. Structural and biochemical data show that mutations of some residues at this site influence the binding of SO4(2-) and affect the enzymatic activity. The results in the present paper suggest that this new SO4(2-)-binding site is a second allosteric site to regulate the enzymatic activity which might also exist in other eukaryotic PRSs (except plant PRSs of class II), but not in bacterial PRSs.