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The selection of correct metal ions with high fidelity against competing cellular cations is crucial for the function of many metalloenzymes; however, the understanding of the principles that govern metal selectivity is still incomplete. In this study, the crystal structure of the Tm1162 protein from Thermotoga maritima, a metallo-β-lactamase, is reported. Several crystal structures of wild-type Tm1162 and its mutants were solved. Homologues of Tm1162 are widely distributed in bacteria and archaea, including several human pathogens. The monomer possesses an αβ/βα fold, with the core β-strands having the β-sheet sandwich structure common to the metallo-β-lactamase superfamily. Tm1162 exists as a trimer in the crystal and this trimeric unit is likely to be present in solution. In the trimer, three active sites reside at the interface between subunits, suggesting that the oligomeric assembly is crucial for catalysis. A new type of structurally encoded heterodinuclear site has been identified by confirming the identity of nickel-containing heteronuclear sites in Tm1162 via X-ray absorption spectroscopy and anomalous difference Fourier maps. The second coordination sphere, including His8 and Glu73, maintains the side-chain orientations of histidines and stabilizes the metal-binding site. Nickel coordination was crucial for the oligomerization of Tm1162. The nickel-dependent and manganese-dependent β-lactamase and phosphodiesterase activities of Tm1162 have also been characterized. [ABSTRACT FROM AUTHOR]

The structures of two mutants (H192A and Y246F) of a mannuronate-specific alginate lyase, A1-III, from Sphingomonas species A1 complexed with a tetrasaccharide substrate [4-deoxy- l- erythro-hex-4-ene-pyranosyluronate-(mannuronate)2-mannuronic acid] were determined by X-ray crystallography at around 2.2 Å resolution together with the apo form of the H192A mutant. The final models of the complex forms, which comprised two monomers (of 353 amino-acid residues each), 268-287 water molecules and two tetrasaccharide substrates, had R factors of around 0.17. A large conformational change occurred in the position of the lid loop (residues 64-85) in holo H192A and Y246F compared with that in apo H192A. The lid loop migrated about 14 Å from an open form to a closed form to interact with the bound tetrasaccharide and a catalytic residue. The tetrasaccharide was bound in the active cleft at subsites −3 to +1 as a substrate form in which the glycosidic linkage to be cleaved existed between subsites −1 and +1. In particular, the Oη atom of Tyr68 in the closed lid loop forms a hydrogen bond to the side chain of a presumed catalytic residue, Oη of Tyr246, which acts both as an acid and a base catalyst in a syn mechanism. [ABSTRACT FROM AUTHOR]

The 8S globulins of mung bean [ Vigna radiata (L.) Wilczek] are vicilin-type seed storage globulins which consist of three isoforms: 8Sα, 8Sα′ and 8Sβ. The three isoforms have high sequence identities with each other (around 90%). The structure of 8Sα globulin has been determined for the first time by X-ray crystallographic analysis and refined at 2.65 Å resolution with a final R factor of 19.6% for 10–2.65 Å resolution data. The refined 8Sα globulin structure consisted of 366 of the 423 amino-acid residues (one subunit of the biological trimer). With the exception of several disordered regions, the overall 8Sα globulin structure closely resembled those of other seed storage 7S globulins. The 8Sα globulin exhibited the highest degree of sequence identity (68%) and structural similarity (a root-mean-square deviation of 0.6 Å) with soybean β-conglycinin β (7S globulin). Their surface hydrophobicities are also similar to each other, although their solubilities differ under alkaline conditions at low ionic strength. This difference seems to be a consequence of charge–charge interactions and not hydrophobic interactions of the surfaces, based on a comparison of the electrostatic potentials of the molecular surfaces. The thermal stability of 8Sα globulin is lower than that of soybean β-conglycinin β. This correlates with the cavity size derived from the crystal structure, although other structural features also have a small effect on the protein's thermal stability. [ABSTRACT FROM AUTHOR]

Transferrin, well known as an iron-transport protein, can bind other metal ions, including toxic ones, and is considered to play an important role in the transportation of such metal ions. Here, a crystal structure of aluminium-bound transferrin is described for the first time. Colourless needle-shaped crystals of aluminium-bound ovotransferrin were obtained in PEG 400 solution. Structural determination was performed by molecular replacement using diferric (iron-bound) ovotransferrin as a model and the structural refinement was performed in the 50–2.15 Å resolution range. The overall organization of the aluminium-bound form is almost the same as the iron-bound form: the protein is folded into two homologous lobes (N- and C-lobes) with two domains; two metal-binding sites are located within the inter-domain clefts of each lobe. Four residues (one Asp, two Tyr and one His) and one bicarbonate anion were found to bind an aluminium ion in either lobe, as in the iron-bound form. The highly similar domain-closed structure of the Al3+-bound form may permit the binding of Al3+-bound transferrin to the transferrin receptor. An unusual interaction, the dilysine trigger, which facilitates iron release at low pH in the endosome, was also found in the Al3+-bound form. These findings support the participation of transferrin in the transport of Al3+ ions in vivo. [ABSTRACT FROM AUTHOR]

Studies the structure of the core region of the soybean Β-conglycinin α′ subunit. Thermal stability of the beta subunit higher than that of the alpha subunit; Thermal stabilities conferred by highly homologous core regions; Cluster of charged residues at the intermonomer interface; Hydrophic solvent-accessible surface.

Inorganic polyphosphate [poly(P)]/ATP-glucomannokinase from Arthrobacter sp. Strain KM phosphorylates glucose and mannose, utilizing both ATP and poly(P) as phosphoryl donors. The enzyme was overexpressed in Escherichia coli, purified and crystallized by means of the hanging-drop vapour-diffusion method with ammonium sulfate as the precipitant. The crystals were3 orthorhombic and belonged to space group P2[SUB1]2[SUB1]2[SUB1], with unit-cell parameters a = 66.2, b = 83.7, c = 103.8Å. Assuming two molecules per asymmetric unit, V[SUBsol] is 0.49. X-ray diffraction data to 2.83Å have been collected from a single crystal. [ABSTRACT FROM AUTHOR]

Imidase is an enzyme, also known as dihydropyrimidinase (EC 3.5.2.2), hydantoinase, dihydropyrimidine hydrase or dihydropyrimidine amidohydrolase, that catalyzes the reversible hydrolysis of 5,6-dihydrouracil to 3-ureidopropionate and many other imides. Substrate specificity, metal contem and amino-acid sequence all differ significantly between bacterial and mammalian imide-hydrolyzing enzymes. In this study, a thermophilic imidase was isolated from pig liver and crystallized. Two kinds of imidase crystals were grown by the hanging-drop vapour-diffusion method using polyethylene glycol MME 5000 and 2-propanol as precipitants. One belongs to the triclinic P[SUB1] space group, with unit-cell parameters a = 9635, b = 96.87. c = 154.87 Å, a = 82.10, β = 72.54, γ = 77.19°, and the other belongs to the orthorhombic C222[SUB1] space group, with unit-cell parameters a = 113.92, b = 157.22, c = 156.21 Å. [ABSTRACT FROM AUTHOR]

Pyridoxal 4-dehydrogenase (PLDH; EC L1.107) is the second enzyme in the bacterial degradation pathway I of vitamin B6, which catalyzes the oxidation of pyridoxal to 4-pyridoxolactone using NAD+. PLDH from Microbacterium luteolum, a dimeric protein with a subunit molecular weight of 38 kDa, was crystallized at 277 K in a drop solution comprising 15%(w/v) polyethylene glycol 4000, 0.15 M sodium acetate, 7.5 mM n-octyl-β-D-glucoside and 0.075 M Tris-HCl pH 7.5 by the sitting-drop vapour-diffusion method. The crystals were monoclinic and belonged to space group C2, with unit-cell parameters a = 107.0, h = 56.7, c = 130.2 Å, β = 103.6°. Diffraction data were collected from a single crystal to 2.0 Å. [ABSTRACT FROM AUTHOR]

Monodehydroascorbate (MDA) radical reductase (EC 1.6.5.4) is an FAD enzyme that catalyzes the univalent reduction of MDA radical to ascorbate using NAD(P)H as an electron donor. The recombinant MDA reductase from cucumber was crystallized using polyethylene glycol 6000 as a precipitant. The crystals belong to space group P21, with unit-cell parameters a = 60.8, b = 138.6, c = 61.7 Å, β = 114.5°, and contained two molecules per asymmetric unit. The Matthews coefficient (VM ) and the solvent content are 2.46 ų Da-1 and 50.0%, respectively. Diffraction data were collected to a resolution of 2.4 Å at 100 K using Cu Kα radiation with a multi-wire area detector and gave a data set with an overall Rsym of 10.0% and a completeness of 92.5%. [ABSTRACT FROM AUTHOR]

Determines the crystallization and X-ray analysis of cold-active protein-tyrosine phosphatase of Shewanella species. Catalytic residue of the enzyme; Measurement of the dimensions of protein crystals; Structural properties of the phosphatase.

NAD kinase from Mycobacterium tuberculosis H37Rv utilizes ATP or inorganic polyphosphate [poly(P)] as a phosphoryl donor for the phosphorylation of NAD. The enzyme overexpressed in Escherichia coli was purified and crystallized by means of the hanging-drop vapour-diffusion method with polyethylene glycol 4000 as the precipitant. Preliminary X-ray analysis of the resultant crystals revealed they belonged to the monoclinic space group C2 and had unit-cell parameters a = 140.0, b = 69.3, c = 106.3 Å β = 130.1°. The molecular weight of the NAD kinase is 35 kDa; assuming that a crystal contains two subunits of the NAD kinase in an asymmetric unit, the solvent content Vsol is 0.62. X-ray diffraction data to 2.99 Å have been collected from the native crystal. [ABSTRACT FROM AUTHOR]

Sphingomonas sp. A1 possesses a macromolecule (alginate; average molecular size 25 700 Da) uptake system mediated by a novel pit-dependent ABC transporter. In this system, AlgS (363 amino-acid residues; 40 kDa) functions as an ATPase and provides energy for the translocation of high molecular-weight alginate across the cytoplasmic membrane. Hexahistidine-tagged AlgS of Sphingomonas sp. Al was overexpressed in Escherichia coli and crystallized by means of the hanging-drop vapour-diffusion method with ammonium dihydrogen monophosphate as the precipitant. Preliminary X-ray analysis of the resultant crystals was performed; they belonged to the monoclinic space group P21 and had unit-cell parameters a = 57.4, b = 92.7, c = 65.8 Å, β = 102.3°. X-ray diffraction data to 3.2 Å have been collected from the native crystal. [ABSTRACT FROM AUTHOR]