Multiwavelength Anomalous Diffraction Method Research Articles

Crystal structure of glutathione-independent formaldehyde dehydrogenase

Formaldehyde dehydrogenase from Pseudomonas putida (PFDH) is a member of the zinc-containing medium-chain alcohol dehydrogenase (ADH) family. The pyridine nucleotide NAD(H) in PFDH, which is distinct from the coenzyme (as co-substrate) in typical ADHs, is tightly but not covalently bound to the protein and acts as a cofactor. Such enzymes with tightly bound NAD(P)(H) acting as a cofactor are called nicotinoproteins. The structural basis of the tightly bound cofactor of PFDH is unknown. The crystal structure of PFDH has been solved by the multiwavelength anomalous diffraction method using intrinsic zinc ions and has been refined at a 1.65 Å resolution. The 170-kDa-homotetrameric PFDH molecule shows 222-point group symmetry. Although the secondary structure arrangement and the binding mode of catalytic and structural zinc ions in PFDH are similar to those of typical ADHs, a number of loop structures that differ between PFDH and ADHs in their lengths and conformations are observed.

Crystal structure of lipocalin-type prostaglandin D synthase

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is responsible for the biosynthesis of PGD 2, a potent endogenous sleep-inducing substance found in the central nervous system. In this study, we crystallized recombinant mouse L-PGDS from a sodium citrate solution by the hanging drop vapor-diffusion method. The crystal structure of the selenomethionyl Cys 65Ala-substituted L-PGDS was determined at 2.5 Å resolution by using the multiwavelength anomalous diffraction method. The crystals belonged to the orthorhombic space group C222 1 with lattice constants a=45.5, b=66.8, c=104.5 Å. The overall architecture of L-PGDS had an eight-stranded β-barrel with a hydrophobic cavity, in which the Cys 65 residue, an active site for the catalysis, was identified.

Crystal Structure of Formaldehyde Dehydrogenase from Pseudomonas putida: the Structural Origin of the Tightly Bound Cofactor in Nicotinoprotein Dehydrogenases

Formaldehyde dehydrogenase from Pseudomonas putida (PFDH) is a member of the zinc-containing medium-chain alcohol dehydrogenase family. The pyridine nucleotide NAD(H) in PFDH, which is distinct from the coenzyme (as cosubstrate) in typical alcohol dehydrogenases (ADHs), is tightly but not covalently bound to the protein and acts as a cofactor. PFDH can catalyze aldehyde dismutations without an external addition of NAD(H). The structural basis of the tightly bound cofactor of PFDH is unknown. The crystal structure of PFDH has been solved by the multiwavelength anomalous diffraction method using intrinsic zinc ions and has been refined at a 1.65Å resolution. The 170-kDa homotetrameric PFDH molecule shows 222 point group symmetry. Although the secondary structure arrangement and the binding mode of catalytic and structural zinc ions in PFDH are similar to those of typical ADHs, a number of loop structures that differ between PFDH and ADHs in their lengths and conformations are observed. A comparison of the present structure of PFDH with that of horse liver ADH, a typical example of an ADH, reveals that a long insertion loop of PFDH shields the adenine part of the bound NAD+ molecule from the solvent, and a tight hydrogen bond network exists between the insertion loop and the adenine part of the cofactor, which is unique to PFDH. This insertion loop is conserved completely among the aldehyde-dismutating formaldehyde dehydrogenases, whereas it is replaced by a short turn among typical ADHs. Thus, the insertion loop specifically found among the aldehyde-dismutating formaldehyde dehydrogenases is responsible for the tight cofactor binding of these enzymes and explains why PFDH can effectively catalyze alternate oxidation and reduction of aldehydes without the release of cofactor molecule from the enzyme.

Structural and functional analysis of enzymes and their application to clinical analysis--study on Pseudomonas putida formaldehyde dehydrogenase

Formaldehyde dehydrogenase (PFDH) was isolated from the creatinine-decomposing bacterium Pseudomonas putida, and its gene has been cloned. PFDH is unique because it was the only enzyme that catalyzed the dehydrogenation of formaldehyde without glutathione. PFDH belongs to a zinc-containing alcohol dehydrogenase family. Quantitative analysis of the reaction products using NMR revealed that the enzyme is not simply a dehydrogenase but is an aldehyde dismutase catalyzing a simultaneous conversion of both aldehyde to carboxylate and aldehyde to alcohol. The enzyme contains a tightly bound cofactor of NAD+/NADH per subunit and is classified as a nicotinoprotein. The enzyme reaction can proceed without external addition of the nucleotide cofactor. The formaldehyde was crystallized using the hanging-drop vapor diffusion method with ammonium sulfate as a precipitant. The crystal structure was determined using the multiwavelength anomalous diffraction method with intrinsic zinc ions. The overall structure of PFDH is similar to that of a classic horse liver alcohol dehydrogenase. However, a comparison of these structures indicated that the insertion loop specifically found in PFDH may be responsible for the tight binding of the cofactor, thereby making PFDH a dismutase.

X-ray crystallographic studies of serotonin N-acetyltransferase catalysis and inhibition

The structure of serotonin N-acetyltransferase (also known as arylalkylamine N-acetyltransferase; AANAT) bound to a potent bisubstrate analog inhibitor has been determined at 2.0 Å resolution using a two-edge (Se, Br) multiwavelength anomalous diffraction (MAD) experiment. This acetyl-CoA dependent enzyme is a member of the GCN5-related family of N-acetyltransferases (GNATs), which share four conserved sequence motifs (A-D). In serotonin N-acetyltransferase, motif A adopts an α/β conformation characteristic of the phylogenetically invariant cofactor binding site seen in all previously characterized GNATs. Motif B displays a significantly lower level of conservation among family members, giving rise to a novel α/β structure for the serotonin binding slot. Utilization of a brominated CoA- S-acetyl-tryptamine-bisubstrate analog inhibitor and the MAD method permitted conclusive identification of two radically different conformations for the tryptamine moiety in the catalytic site ( cis and trans). A second high-resolution X-ray structure of the enzyme bound to a bisubstrate analog inhibitor, with a longer tether between the acetyl-CoA and tryptamine moieties, demonstrates only the trans conformation. Given a previous proposal that AANAT can catalyze an alkyltransferase reaction in a conformationally altered active site relative to its acetyltransferase activity, it is possible that the two conformations of the bisubstrate analog observed crystallographically correspond to these alternative reaction pathways. Our findings may ultimately lead to the design of analogs with improved AANAT inhibitory properties for in vivo applications.

Crystal Structure of a Novel Carboxypeptidase from the Hyperthermophilic Archaeon Pyrococcus furiosus

The structure of Pyrococcus furiosus carboxypeptidase (PfuCP) has been determined to 2.2 Å resolution using multiwavelength anomalous diffraction (MAD) methods. PfuCP represents the first structure of the new M32 family of carboxypeptidases. The overall structure is comprised of a homodimer. Each subunit is mostly helical with its most pronounced feature being a deep substrate binding groove. The active site lies at the bottom of this groove and contains an HEXXH motif that coordinates the metal ion required for catalysis. Surprisingly, the structure is similar to the recently reported rat neurolysin. Comparison of these structures as well as sequence analyses with other homologous proteins reveal several conserved residues. The roles for these conserved residues in the catalytic mechanism are inferred based on modeling and their location.

Crystal structure of an RNA helix recognized by a zinc-finger protein: an 18-bp duplex at 1.6 A resolution.

The crystal structure of the 19-mer RNA, 5'-GAAUGCCUGCGAGCAUCCC-3' has been determined from X-ray diffraction data to 1.6 A resolution by the multiwavelength anomalous diffraction method from crystals containing a brominated uridine. In the crystal, this RNA forms an 18-mer self-complementary double helix with the 19th nucleotide flipped out of the helix. This helix contains most of the target stem recognized by the bacteriophage Mu Com protein (control of mom), which activates translation of an unusual DNA modification enzyme, Mom. The 19-mer duplex, which contains one A.C mismatch and one A.C/G.U tandem wobble pair, was shown to bind to the Com protein by native gel electrophoresis shift assay. Comparison of the geometries and base stacking properties between Watson-Crick base pairs and the mismatches in the crystal structure suggest that both hydrogen bonding and base stacking are important for stabilizing these mismatched base pairs, and that the unusual geometry adopted by the A.C mismatch may reveal a unique structural motif required for the function of Com.

Multiwavelength anomalous diffraction analysis at the M absorption edges of uranium.

The multiwavelength anomalous diffraction (MAD) method for phase evaluation is now widely used in macromolecular crystallography. Successful MAD structure determinations have been carried out at the K or L absorption edges of a variety of elements. In this study, we investigate the anomalous scattering properties of uranium at its M(IV) (3.326 A) and M(V) (3.490 A) edge. Fluorescence spectra showed remarkably strong anomalous scattering at these edges (f' = -70e, f" = 80e at the M(IV) edge and f' = -90e, f" = 105e at the M(V) edge), many times higher than from any anomalous scatterers used previously for MAD phasing. However, the large scattering angles and high absorption at the low energies of these edges present some difficulties not found in typical crystallographic studies. We conducted test experiments at the M(IV) edge with crystals of porcine elastase derivatized with uranyl nitrate. A four-wavelength MAD data set complete to 3.2-A Bragg spacings was collected from a single small frozen crystal. Analysis of the data yielded satisfactory phase information (average difference of (0)phi(T) - (0)phi(A) for replicated determinations is 32 degrees ) and produced an interpretable electron-density map. Our results demonstrate that it is practical to measure macromolecular diffraction data at these edges with current instrumentation and that phase information of good accuracy can be extracted from such experiments. We show that such experiments have potential for the phasing of very large macromolecular assemblages.

The κ-carrageenase of P. carrageenovora Features a Tunnel-Shaped Active Site : A Novel Insight in the Evolution of Clan-B Glycoside Hydrolases

Background: κ-carrageenans are gel-forming, sulfated 1,3-α-1,4-β-galactans from the cell walls of marine red algae. The κ-carrageenase from the marine, gram-negative bacterium Pseudoalteromonas carrageenovora degrades κ-carrageenan both in solution and in solid state by an endoprocessive mechanism. This β-galactanase belongs to the clan-B of glycoside hydrolases. Results: The structure of P. carrageenovora κ-carrageenase has been solved to 1.54 Å resolution by the multiwavelength anomalous diffraction (MAD) method, using a seleno-methionine-substituted form of the enzyme. The enzyme folds into a curved β sandwich, with a tunnel-like active site cavity. Another remarkable characteristic is the presence of an arginine residue at subsite −1. Conclusions: The crystal structure of P. carrageenovora κ-carrageenase is the first three-dimensional structure of a carrageenase. Its tunnel-shaped active site, the first to be reported for enzymes other than cellulases, suggests that such tunnels are associated with the degradation of solid polysaccharides. Clan-B glycoside hydrolases fall into two subgroups, one with catalytic machinery held by an ancestral β bulge, and the other in which it is held by a regular β strand. At subsite −1, all of these hydrolases exhibit an aromatic amino acid that interacts with the hexopyranose ring of the monosaccharide undergoing catalysis. In addition, in κ-carrageenases, an arginine residue recognizes the sulfate-ester substituents of the β-linked κ-carrageenan monomers. It also appears that, in addition to the nucleophile and acid/base catalysts, two other amino acids are involved with the catalytic cycle, accelerating the deglycosylation step.

Crystal Structure of Riboflavin Synthase

Background: Riboflavin synthase catalyzes the dismutation of two molecules of 6,7-dimethyl-8-(1′-D-ribityl)-lumazine to yield riboflavin and 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine. The homotrimer of 23 kDa subunits has no cofactor requirements for catalysis. The enzyme is nonexistent in humans and is an attractive target for antimicrobial agents of organisms whose pathogenicity depends on their ability to biosynthesize riboflavin. Results: The first three-dimensional structure of the enzyme was determined at 2.0 Å resolution using the multiwavelength anomalous diffraction (MAD) method on the Escherichia coli protein containing selenomethionine residues. The homotrimer consists of an asymmetric assembly of monomers, each of which comprises two similar β barrels and a C-terminal α helix. The similar β barrels within the monomer confirm a prediction of pseudo two-fold symmetry that is inferred from the sequence similarity between the two halves of the protein. The β barrels closely resemble folds found in phthalate dioxygenase reductase and other flavoproteins. Conclusions: The three active sites of the trimer are proposed to lie between pairs of monomers in which residues conserved among species reside, including two Asp-His-Ser triads and dyads of Cys-Ser and His-Thr. The proposed active sites are located where FMN (an analog of riboflavin) is modeled from an overlay of the β barrels of phthalate dioxygenase reductase and riboflavin synthase. In the trimer, one active site is formed, and the other two active sites are wide open and exposed to solvent. The nature of the trimer configuration suggests that only one active site can be formed and be catalytically competent at a time.

Crystal Structure of 3,4-Dihydroxy-2-Butanone 4-Phosphate Synthase of Riboflavin Biosynthesis

Background: 3,4-Dihydroxy-2-butanone-4-phosphate synthase catalyzes a commitment step in the biosynthesis of riboflavin. On the enzyme, ribulose 5-phosphate is converted to 3,4-dihydroxy-2-butanone 4-phosphate and formate in steps involving enolization, ketonization, dehydration, skeleton rearrangement, and formate elimination. The enzyme is absent in humans and an attractive target for the discovery of antimicrobials for pathogens incapable of acquiring sufficient riboflavin from their hosts. The homodimer of 23 kDa subunits requires Mg 2+ for activity. Results: The first three-dimensional structure of the enzyme was determined at 1.4 Å resolution using the multiwavelength anomalous diffraction (MAD) method on Escherichia coli protein crystals containing gold. The protein consists of an α + β fold having a complex linkage of β strands. Intersubunit contacts are mediated by numerous hydrophobic interactions and three hydrogen bond networks. Conclusions: A proposed active site was identified on the basis of amino acid residues that are conserved among the enzyme from 19 species. There are two well-separated active sites per dimer, each of which comprise residues from both subunits. In addition to three arginines and two threonines, which may be used for recognizing the phosphate group of the substrate, the active site consists of three glutamates, two aspartates, two histidines, and a cysteine which may provide the means for general acid and base catalysis and for coordinating the Mg 2+ cofactor within the active site.

Single-wavelength anomalous diffraction phasing revisited.

Multiwavelength anomalous diffraction (MAD) phasing has become a routinely used tool for determining new macromolecular structures. The MAD method has stringent data-collection requirements, typically necessitating radiation-resistant crystals and access to a tunable synchrotron beamline. In cases where synchrotron time, monochromator tunability or radiation damage is a concern or where high-throughput structure determination is desired, phasing methods capable of producing interpretable electron-density maps from less data become attractive alternatives to MAD. The increasing availability of tunable synchrotron data-collection facilities prompted the authors to revisit single-wavelength anomalous diffraction (SAD) phasing used in conjunction with a phase-ambiguity resolving method such as solvent flattening. The anomalous diffraction from seven different selenomethionine-labelled protein crystals has been analysed and it is shown that in conjunction with solvent flattening, diffraction data from the peak anomalous wavelength alone can produce interpretable electron-density maps of comparable quality to those resulting from full MAD phasing. Single-wavelength anomalous diffraction (SAD) phasing can therefore be a time-efficient alternative to MAD. The data also show that radiation damage can have a significant effect on the quality of SAD/MAD diffraction data. These results may be useful in the design of optimal strategies for collection of the diffraction data.

The Structure of Human β-Defensin-2 Shows Evidence of Higher Order Oligomerization

Defensins are small cationic peptides that are crucial components of innate immunity, serving as both antimicrobial agents and chemoattractant molecules. The specific mechanism of antimicrobial activity involves permeabilization of bacterial membranes. It has been postulated that individual monomers oligomerize to form a pore through anionic membranes, although the evidence is only indirect. Here, we report two high resolution x-ray structures of human beta-defensin-2 (hBD2). The phases were experimentally determined by the multiwavelength anomalous diffraction method, utilizing a novel, rapid method of derivatization with halide ions. Although the shape and charge distribution of the monomer are similar to those of other defensins, an additional alpha-helical region makes this protein topologically distinct from the mammalian alpha- and beta-defensin structures reported previously. hBD2 forms dimers topologically distinct from that of human neutrophil peptide-3. The quaternary octameric arrangement of hBD2 is conserved in two crystal forms. These structures provide the first detailed description of dimerization of beta-defensins, and we postulate that the mode of dimerization of hBD2 is representative of other beta-defensins. The structural and electrostatic properties of the hBD2 octamer support an electrostatic charge-based mechanism of membrane permeabilization by beta-defensins, rather than a mechanism based on formation of bilayer-spanning pores.

Observation of an unexpected third receptor molecule in the crystal structure of human interferon-γ receptor complex

Background: Molecular interactions among cytokines and cytokine receptors form the basis of many cell-signaling pathways relevant to immune function. Interferon-γ (IFN-γ) signals through a multimeric receptor complex consisting of two different but structurally related transmembrane chains: the high-affinity receptor-binding subunit (IFN-γRα) and a species-specific accessory factor (AF-1 or IFN-γRβ). In the signaling complex, the two receptors probably interact with one another through their extracellular domains. Understanding the atomic interactions of signaling complexes enhances the ability to control and alter cell signaling and also provides a greater understanding of basic biochemical processes. Results: The crystal structure of the complex of human IFN-γ with the soluble, glycosylated extracellular part of IFN-γRα has been determined at 2.9 Å resolution using multiwavelength anomalous diffraction methods. In addition to the expected 2:1 complex, the crystal structure reveals the presence of a third receptor molecule not directly associated with the IFN-γ dimer. Two distinct intermolecular contacts, involving the edge strands of the C-terminal domains, are observed between this extra receptor and the 2:1 receptor–ligand complex thereby forming a 3:1 complex. Conclusions: The observed interactions in the 2:1 complex of the high-affinity cell-surface receptor with the IFN-γ cytokine are similar to those seen in a previously reported structure where the receptor chains were not glycosylated. The formation of β-sheet packing interactions between pairs of IFN-γRα receptors in these crystals suggests a possible model for receptor oligomerization of Rα and the structurally homologous Rβ receptors in the fully active IFN-γ signaling complex.

Crystal structure of the pyridoxal 5'-phosphate dependent L-methionine gamma-lyase from Pseudomonas putida.

L-Methionine gamma-lyase (MGL) catalyzes the pyridoxal 5'-phosphate (PLP) dependent alpha,gamma-elimination of L-methionine. We have determined two crystal structures of MGL from Pseudomonas putida using MAD (multiwavelength anomalous diffraction) and molecular replacement methods. The structures have been refined to an R-factor of 21.1% at 2.0 and 1.7 A resolution using synchrotron radiation diffraction data. A homotetramer with 222 symmetry is built up by non-crystallographic symmetry. Two monomers associate to build the active dimer. The spatial fold of subunits, with three functionally distinct domains and their quarternary arrangement, is similar to those of L-cystathionine beta-lyase and L-cystathionine gamma-synthase from Escherichia coli.

Crystal structure of a methyltetrahydrofolate- and corrinoid-dependent methyltransferase

Background: Methyltetrahydrofolate, corrinoid iron–sulfur protein methyltransferase (MeTr), catalyzes a key step in the Wood-Ljungdahl pathway of carbon dioxide fixation. It transfers the N 5-methyl group from methyltetrahydrofolate (CH 3-H 4folate) to a cob(I)amide center in another protein, the corrinoid iron–sulfur protein. MeTr is a member of a family of proteins that includes methionine synthase and methanogenic enzymes that activate the methyl group of methyltetra-hydromethano(or-sarcino)pterin. We report the first structure of a protein in this family. Results: We determined the crystal structure of MeTr from Clostridium thermoaceticum at 2.2 Å resolution using multiwavelength anomalous diffraction methods. The overall architecture presents a new functional class of the versatile triose phosphate isomerase (TIM) barrel fold. The MeTr tertiary structure is surprisingly similar to the crystal structures of dihydropteroate synthetases despite sharing less than 20% sequence identity. This homology permitted the methyl-H 4folate binding site to be modeled. The model suggests extensive conservation of the pterin ring binding residues in the polar active sites of the methyltransferases and dihydropteroate synthetases. The most significant structural difference between these enzymes is in a loop structure above the active site. It is quite open in MeTr, where it can be modeled as the cobalamin binding site. Conclusions: The MeTr structure consists of a TIM barrel that embeds methyl-H 4folate and cobamide. All related methyltransferases are predicted to fold into a similar TIM barrel pattern and have a similar pterin and cobamide binding site. The observed structure is consistent with either a ‘front’ (N 5) or ‘back’ (C 8a) side protonation of CH 3-H 4folate, a key step that enhances the electrophilic character of the methyl group, activating it for nucleophilic attack by Co(I).

Use of CrKα radiation to enhance the signal from anomalous scatterers including sulfur

The anomalous signals from scatterers such as sulfur (S) and arsenic (As) were compared in diffraction data sets collected from an X-ray source with three different targets, Au, Cu and Cr, on a multi-target rotating anode. HIV-1 integrase crystals served as the test case for this study. The crystalline specimen of HIV-1 integrase contains in each protein molecule two As atoms, each covalently bound to a cysteine S atom, and two additional S atoms derived from methionine. It was found that the CrKα radiation gave the clearest peaks in anomalous difference Fourier maps, although the signal-to-noise ratios of the anomalous signal for the CuKα and CrKα data were similar but better than that for AuLα. This result was in spite of the fourfold higher flux from the Cu anodeversusthe Cr anode. For all three X-ray wavelengths, anomalous difference Fourier maps calculated with bias-removed phases derived from the known atomic model revealed clear peaks at the two As sites. However, only in the map calculated using the CrKα data were both peaks of the expected ellipsoidal shape, enveloping the As atom and the adjacent S atom. None of the S sites was apparent in difference maps calculated using the AuLα data. The ability to enhance the S-derived anomalous signal using CrKα radiation has particularly useful applications in the structure determination of proteins, for example in resolving ambiguities in the chain tracing of a protein with numerous disulfide bonds and in assigning amino acid identities. Additionally, anomalous difference Patterson maps calculated from the CrKα data were sufficiently clear to identify the As-related peaks. These results form the groundwork for in-house phase determination with the multi-wavelength anomalous diffraction method.

Structure of cyanase reveals that a novel dimeric and decameric arrangement of subunits is required for formation of the enzyme active site

Background: Cyanase is an enzyme found in bacteria and plants that catalyzes the reaction of cyanate with bicarbonate to produce ammonia and carbon dioxide. In Escherichia coli, cyanase is induced from the cyn operon in response to extracellular cyanate. The enzyme is functionally active as a homodecamer of 17 kDa subunits, and displays half-site binding of substrates or substrate analogs. The enzyme shows no significant amino acid sequence homology with other proteins. Results: We have determined the crystal structure of cyanase at 1.65 Å resolution using the multiwavelength anomalous diffraction (MAD) method. Cyanase crystals are triclinic and contain one homodecamer in the asymmetric unit. Selenomethionine-labeled protein offers 40 selenium atoms for use in phasing. Structures of cyanase with bound chloride or oxalate anions, inhibitors of the enzyme, allowed identification of the active site. Conclusions: The cyanase monomer is composed of two domains. The N-terminal domain shows structural similarity to the DNA-binding α-helix bundle motif. The C-terminal domain has an ‘open fold’ with no structural homology to other proteins. The subunits of cyanase are arranged in a novel manner both at the dimer and decamer level. The dimer structure reveals the C-terminal domains to be intertwined, and the decamer is formed by a pentamer of these dimers. The active site of the enzyme is located between dimers and is comprised of residues from four adjacent subunits of the homodecamer. The structural data allow a conceivable reaction mechanism to be proposed.

Crystal structure of NAD(+)-dependent DNA ligase: modular architecture and functional implications.

DNA ligases catalyze the crucial step of joining the breaks in duplex DNA during DNA replication, repair and recombination, utilizing either ATP or NAD(+) as a cofactor. Despite the difference in cofactor specificity and limited overall sequence similarity, the two classes of DNA ligase share basically the same catalytic mechanism. In this study, the crystal structure of an NAD(+)-dependent DNA ligase from Thermus filiformis, a 667 residue multidomain protein, has been determined by the multiwavelength anomalous diffraction (MAD) method. It reveals highly modular architecture and a unique circular arrangement of its four distinct domains. It also provides clues for protein flexibility and DNA-binding sites. A model for the multidomain ligase action involving large conformational changes is proposed.

2C1615 硫酸還元菌由来高分子量チトクロムcのX線結晶構造解析

2C1615 硫酸還元菌由来高分子量チトクロムcのX線結晶構造解析