Selenium Sites Research Articles

Structure of Chlorobium tepidum Sepiapterin Reductase Complex Reveals the Novel Substrate Binding Mode for Stereospecific Production of l-threo-Tetrahydrobiopterin

Sepiapterin reductase (SR) is involved in the last step of tetrahydrobiopterin (BH(4)) biosynthesis by reducing the di-keto group of 6-pyruvoyl tetrahydropterin. Chlorobium tepidum SR (cSR) generates a distinct BH(4) product, L-threo-BH(4) (6R-(1'S,2'S)-5,6,7,8-BH(4)), whereas animal enzymes produce L-erythro-BH(4) (6R-(1'R,2'S)-5,6,7,8-BH(4)) although it has high amino acid sequence similarities to the other animal enzymes. To elucidate the structural basis for the different reaction stereospecificities, we have determined the three-dimensional structures of cSR alone and complexed with NADP and sepiapterin at 2.1 and 1.7 A resolution, respectively. The overall folding of the cSR, the binding site for the cofactor NADP(H), and the positions of active site residues were quite similar to the mouse and the human SR. However, significant differences were found in the substrate binding region of the cSR. In comparison to the mouse SR complex, the sepiapterin in the cSR is rotated about 180 degrees around the active site and bound between two aromatic side chains of Trp-196 and Phe-99 so that its pterin ring is shifted to the opposite side, but its side chain position is not changed. The swiveled sepiapterin binding results in the conversion of the side chain configuration, exposing the opposite face for hydride transfer from NADPH. The different sepiapterin binding mode within the conserved catalytic architecture presents a novel strategy of switching the reaction stereospecificities in the same protein fold.

Crystal structure of alanyl‐tRNA synthetase editing‐domain homolog (PH0574) from a hyperthermophile, Pyrococcus horikoshii OT3 at 1.45 Å resolution

Crystal structure of alanyl‐tRNA synthetase editing‐domain homolog (PH0574) from a hyperthermophile, <i>Pyrococcus horikoshii</i> OT3 at 1.45 Å resolution

Structural and Functional Evidence for Bacillus subtilis PaiA as a Novel N1-Spermidine/Spermine Acetyltransferase

Bacillus subtilis PaiA has been implicated in the negative control of sporulation as well as production of degradative enzymes. PaiA shares recognizable sequence homology with N-acetyltransferases, including those that can acetylate spermidine/spermine substrates. We have determined the crystal structure of PaiA in complex with CoA at 1.9 A resolution and found that PaiA is a member of the N-acetyltransferase superfamily of enzymes. Unexpectedly, we observed the binding of an oxidized CoA dimer in the active site of PaiA, and the structural information suggests the substrates of the enzyme could be linear, positively charged compounds. Our biochemical characterization is also consistent with this possibility, since purified PaiA possesses N1-acetyltransferase activity toward polyamine substrates including spermidine and spermine. Further, conditional overexpression of PaiA in bacteria results in increased acetylation of endogenous spermidine pools. Thus, our structural and biochemical analyses indicate that PaiA is a novel N-acetyltransferase capable of acetylating both spermidine and spermine. In this way, the pai operon may function in regulating intracellular polyamine concentrations and/or binding capabilities. In addition to preventing toxicity due to polyamine excess, this function may also serve to regulate expression of certain bacterial gene products such as those involved in sporulation.

GTP Cyclohydrolase II Structure and Mechanism

GTP cyclohydrolase II converts GTP to 2,5-diamino-6-beta-ribosyl-4(3H)-pyrimidinone 5'-phosphate, formate and pyrophosphate, the first step in riboflavin biosynthesis. The essential role of riboflavin in metabolism and the absence of GTP cyclohydrolase II in higher eukaryotes makes it a potential novel selective antimicrobial drug target. GTP cyclohydrolase II catalyzes a distinctive overall reaction from GTP cyclohydrolase I; the latter converts GTP to dihydroneopterin triphosphate, utilized in folate and tetrahydrobiopterin biosynthesis. The structure of GTP cyclohydrolase II determined at 1.54-A resolution reveals both a different protein fold to GTP cyclohydrolase I and distinctive molecular recognition determinants for GTP; although in both enzymes there is a bound catalytic zinc. The GTP cyclohydrolase II.GMPCPP complex structure shows Arg(128) interacting with the alpha-phosphonate, and thus in the case of GTP, Arg(128) is positioned to act as the nucleophile for pyrophosphate release and formation of the proposed covalent guanylyl-GTP cyclohydrolase II intermediate. Tyr(105) is identified as playing a key role in GTP ring opening; it is hydrogen-bonded to the zinc-activated water molecule, the latter being positioned for nucleophilic attack on the guanine C-8 atom. Although GTP cyclohydrolase I and GTP cyclohydrolase II both use a zinc ion for the GTP ring opening and formate release, different residues are utilized in each case to catalyze this reaction step.

Activation of the Hetero-octameric ATP Phosphoribosyl Transferase through Subunit Interface Rearrangement by a tRNA Synthetase Paralog

ATP phosphoribosyl transferase (ATP-PRT) joins ATP and 5-phosphoribosyl-1-pyrophosphate (PRPP) in a highly regulated reaction that initiates histidine biosynthesis. The unusual hetero-octameric version of ATP-PRT includes four HisG(S) catalytic subunits based on the periplasmic binding protein fold and four HisZ regulatory subunits that resemble histidyl-tRNA synthetases. Here, we present the first structure of a PRPP-bound ATP-PRT at 2.9 A and provide a structural model for allosteric activation based on comparisons with other inhibited and activated ATP-PRTs from both the hetero-octameric and hexameric families. The activated state of the octameric enzyme is characterized by an interstitial phosphate ion in the HisZ-HisG interface and new contacts between the HisZ motif 2 loop and the HisG(S) dimer interface. These contacts restructure the interface to recruit conserved residues to the active site, where they activate pyrophosphate to promote catalysis. Additionally, mutational analysis identifies the histidine binding sites within a region highly conserved between HisZ and the functional HisRS. Through the oligomerization and functional re-assignment of protein domains associated with aminoacylation and phosphate binding, the HisZ-HisG octameric ATP-PRT acquired the ability to initiate the synthesis of a key metabolic intermediate in an allosterically regulated fashion.

Structural Basis for Autoinhibition and Mutational Activation of Eukaryotic Initiation Factor 2α Protein Kinase GCN2*[boxs

The GCN2 protein kinase coordinates protein synthesis with levels of amino acid stores by phosphorylating eukaryotic translation initiation factor 2. The autoinhibited form of GCN2 is activated in cells starved of amino acids by binding of uncharged tRNA to a histidyl-tRNA synthetase-like domain. Replacement of Arg-794 with Gly in the PK domain (R794G) activates GCN2 independently of tRNA binding. Crystal structures of the GCN2 protein kinase domain have been determined for wild-type and R794G mutant forms in the apo state and bound to ATP/AMPPNP. These structures reveal that GCN2 autoinhibition results from stabilization of a closed conformation that restricts ATP binding. The R794G mutant shows increased flexibility in the hinge region connecting the N- and C-lobes, resulting from loss of multiple interactions involving Arg794. This conformational change is associated with intradomain movement that enhances ATP binding and hydrolysis. We propose that intramolecular interactions following tRNA binding remodel the hinge region in a manner similar to the mechanism of enzyme activation elicited by the R794G mutation.

Structure of Escherichia coli UMP Kinase Differs from That ofOther Nucleoside Monophosphate Kinases and Sheds New Light on EnzymeRegulation

Structure of Escherichia coli UMP Kinase Differs from That ofOther Nucleoside Monophosphate Kinases and Sheds New Light on EnzymeRegulation

Activation of the LicT Transcriptional Antiterminator Involves a Domain Swing/Lock Mechanism Provoking Massive Structural Changes

The transcriptional antiterminator protein LicT regulates the expression of Bacillus subtilis operons involved in beta-glucoside metabolism. It consists of an N-terminal RNA-binding domain (co-antiterminator (CAT)) and two phosphorylatable phosphotransferase system regulation domains (PRD1 and PRD2). In the activated state, each PRD forms a dimeric unit with the phosphorylation sites totally buried at the dimer interface. Here we present the 1.95 A resolution structure of the inactive LicT PRDs as well as the molecular solution structure of the full-length protein deduced from small angle x-ray scattering. Comparison of native (inactive) and mutant (constitutively active) PRD crystal structures shows massive tertiary and quaternary rearrangements of the entire regulatory domain. In the inactive state, a wide swing movement of PRD2 results in dimer opening and brings the phosphorylation sites to the protein surface. This movement is accompanied by additional structural rearrangements of both the PRD1-PRD1 ' interface and the CAT-PRD1 linker. Small angle x-ray scattering experiments indicate that the amplitude of the PRD2 swing might even be wider in solution than in the crystals. Our results suggest that PRD2 is highly mobile in the native protein, whereas it is locked upon activation by phosphorylation.

Crystal Structure of Vinorine Synthase, the First Representative of the BAHD Superfamily

Vinorine synthase is an acetyltransferase that occupies a central role in the biosynthesis of the antiarrhythmic monoterpenoid indole alkaloid ajmaline in the plant Rauvolfia. Vinorine synthase belongs to the benzylalcohol acetyl-, anthocyanin-O-hydroxy-cinnamoyl-, anthranilate-N-hydroxy-cinnamoyl/benzoyl-, deacetylvindoline acetyltransferase (BAHD) enzyme superfamily, members of which are involved in the biosynthesis of several important drugs, such as morphine, Taxol, or vindoline, a precursor of the anti-cancer drugs vincaleucoblastine and vincristine. The x-ray structure of vinorine synthase is described at 2.6-angstrom resolution. Despite low sequence identity, the two-domain structure of vinorine synthase shows surprising similarity with structures of several CoA-dependent acyltransferases such as dihydrolipoyl transacetylase, polyketide-associated protein A5, and carnitine acetyltransferase. All conserved residues typical for the BAHD family are found in domain 1. His160 of the HXXXD motif functions as a general base during catalysis. It is located in the center of the reaction channel at the interface of both domains and is accessible from both sides. The channel runs through the entire molecule, allowing the substrate and co-substrate to bind independently. Asp164 points away from the catalytic site and seems to be of structural rather than catalytic importance. Surprisingly, the DFGWG motif, which is indispensable for the catalyzed reaction and unique to the BAHD family, is located far away from the active site and seems to play only a structural role. Vinorine synthase represents the first solved protein structure of the BAHD superfamily.

Crystal Structure of the tRNA 3′ Processing Endoribonuclease tRNase Z from Thermotoga maritima

The maturation of the tRNA 3' end is catalyzed by a tRNA 3' processing endoribonuclease named tRNase Z (RNase Z or 3'-tRNase) in eukaryotes, Archaea, and some bacteria. The tRNase Z generally cuts the 3' extra sequence from the precursor tRNA after the discriminator nucleotide. In contrast, Thermotoga maritima tRNase Z cleaves the precursor tRNA precisely after the CCA sequence. In this study, we determined the crystal structure of T. maritima tRNase Z at 2.6-A resolution. The tRNase Z has a four-layer alphabeta/betaalpha sandwich fold, which is classified as a metallo-beta-lactamase fold, and forms a dimer. The active site is located at one edge of the beta-sandwich and is composed of conserved motifs. Based on the structure, we constructed a docking model with the tRNAs that suggests how tRNase Z may recognize the substrate tRNAs.

The Human TREX2 3′ → 5′-Exonuclease Structure Suggests a Mechanism for Efficient Nonprocessive DNA Catalysis

The 3' --> 5'-exonucleases process DNA ends in many DNA repair pathways of human cells. Determination of the human TREX2 structure is the first of a dimeric 3'-deoxyribonuclease and indicates how this highly efficient nonprocessive enzyme removes nucleotides at DNA 3' termini. Symmetry in the TREX2 dimer positions the active sites at opposite outer edges providing open access for the DNA. Adjacent to each active site is a flexible region containing three arginines positioned appropriately to bind DNA and to control its entry into the active site. Mutation of these three arginines to alanines reduces the DNA binding capacity by approximately 100-fold with no effect on catalysis. The human TREX2 catalytic residues overlay with the bacterial DnaQ family of 3'-exonucleases confirming the structural conservation of the catalytic sites despite limited sequence identity, and mutations of these residues decrease the still measurable activity by approximately 10(5)-fold, confirming their catalytic role.

X-ray absorption, refraction and resonant scattering tensors in selenated protein crystals: implications for data collection strategies in macromolecular crystallography

Polarized fluorescence spectra were recorded in the vicinity of the Se and BrKedges on crystals of the selenated protein aldose reductase in complex with a brominated inhibitor molecule. The X-ray absorption, refraction and resonant scattering tensors as a function of X-ray energy were derived from these data. Substantial dichroism and anisotropy of resonant scattering were observed in these crystals. It is predicted that these effects are present in many resonant scattering experiments in macromolecular crystallography and are likely to affect the diffraction data. As a consequence, the anomalous phasing signal in single- or multi-wavelength anomalous diffraction experiments can be optimized simply by choosing a judicious orientation of the crystal with respect to the polarization direction of the incident X-ray beam. A simple procedure is presented to achieve this, prior to any knowledge about the selenium sites.

Crystal structure of TT1662 from Thermus thermophilus HB8: A member of the α/β hydrolase fold enzymes

Crystal structure of TT1662 from <i>Thermus thermophilus</i> HB8: A member of the α/β hydrolase fold enzymes

Crystallization and preliminary X-ray analysis of the coiled-coil domain of dystrophia myotonica kinase

The coiled-coil domain of dystrophia myotonica protein kinase (DMPK) has been cloned, overexpressed, purified and crystallized. Two crystal forms have been obtained that belong to space groups P3 and P2(1)2(1)2(1) and diffract to 2.4 and 1.6 A resolution, respectively. Experimental phases were obtained by MAD from an SeMet derivative. The location of selenium sites used molecular-replacement phases obtained from search models lacking sequence similarity with the coiled-coil under study. Both crystal forms contain three polypeptide chains in the asymmetric unit.

Crystal structure of a conserved hypothetical protein TT1751 from Thermus thermophilus HB8.

Crystal structure of a conserved hypothetical protein TT1751 from Thermus thermophilus HB8.

Crystal structure of pyranose 2-oxidase from the white-rot fungus Peniophora sp.

Pyranose 2-oxidase catalyzes the oxidation of a number of carbohydrates using dioxygen. The enzyme forms a D(2) symmetric homotetramer and contains one covalently bound FAD per subunit. The structure of the enzyme from Peniophora sp. was determined by multiwavelength anomalous diffraction (MAD) based on 96 selenium sites per crystallographic asymmetric unit and subsequently refined to good-quality indices. According to its chain fold, the enzyme belongs to the large glutathione reductase family and, in a more narrow sense, to the glucose-methanol-choline oxidoreductase (GMC) family. The tetramer contains a spacious central cavity from which the substrate enters one of the four active centers by penetrating a mobile barrier. Since this cavity can only be accessed by glucose-sized molecules, the enzyme does not convert sugars that are part of a larger molecule. The geometry of the active center and a comparison with an inhibitor complex of the homologous enzyme cellobiose dehydrogenase allow the modeling of the reaction at a high confidence level.

Crystal structure of hypothetical protein PH0642 from Pyrococcus horikoshii at 1.6A resolution.

Crystal structure of hypothetical protein PH0642 from Pyrococcus horikoshii at 1.6A resolution.

NMR analysis of surfaces and interfaces in 2-nm CdSe.

Solid-state NMR analysis on wurtzite 2-nm hexadecylamine-capped CdSe nanocrystals (CdSe-HDA) provides evidence of discrete nanoparticle reconstruction within the Se sublattice of the nanomaterial. The cadmium and selenium atoms are probed with (1)H-(113)Cd and (1)H-(77)Se cross-polarization magic angle spinning (MAS) experiments, which demonstrate five ordered selenium sites in the nanoparticle that can be assigned to contributions arising from different surface sites and a selenium site one layer down from the surface. Intriguingly, in these materials both HDA and thiophenol are observed to selectively bind to specific sites on the nanoparticle surface. 2D heteronuclear chemical shift correlation (HETCOR) experiments provide evidence for thiophenol selectively binding at surface vacancies. Analysis of the NMR provides a model of a 2-nm CdSe-HDA molecular surface.

Crystal structure of the TM1442 protein from Thermotoga maritima, a homolog of the Bacillus subtilis general stress response anti-anti-sigma factor RsbV.

Crystal structure of the TM1442 protein from Thermotoga maritima, a homolog of the Bacillus subtilis general stress response anti-anti-sigma factor RsbV.

Functional assignment based on structural analysis: crystal structure of the yggJ protein (HI0303) of Haemophilus influenzae reveals an RNA methyltransferase with a deep trefoil knot.

Farhad Forouhar, Jianwei Shen, Rong Xiao, Thomas B. Acton, Gaetano T. Montelione, and Liang Tong* Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, New York Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers University, New Brunswick, New Jersey Department of Biochemistry, Robert Wood Johnson Medical School, Northeast Structural Genomics Consortium, Piscataway, New Jersey