Biomedical subjects

Keith S Wilson

Publications and source records attributed to Keith S Wilson.

29 records · Page 2Linked to original sources

PubMed2004

The crystal structure of a complex of Campylobacter jejuni dUTPase with substrate analogue sheds light on the mechanism and suggests the "basic module" for dimeric d(C/U)TPases.

The crystal structure of the dUTPase from the important gastric pathogen Campylobacter jejuni has been solved at 1.65 A spacing. This essential bacterial enzyme is the second representative of the new family of dimeric dUTPases to be structurally characterised. Members of this family have a novel all-alpha fold and are unrelated to the all-beta dUTPases of the majority of organisms including eukaryotes such as humans, bacteria such as Escherichia coli, archaea like Methanococcus jannaschii and animal viruses. Therefore, dimeric dUTPases can be considered as candidate drug targets. The X-ray structure of the C.jejuni dUTPase in complex with the non-hydrolysable substrate analogue dUpNHp allows us to define the positions of three catalytically significant phosphate-binding magnesium ions and provides a starting point for a detailed understanding of the mechanism of dUTP/dUDP hydrolysis by dimeric dUTPases. Indeed, a water molecule present in the structure is ideally situated to act as the attacking nucleophile during hydrolysis. A comparison of the dUTPases from C.jejuni and Trypanosoma cruzi reveals a common fold with certain distinct features, both in the rigid and mobile domains as defined in the T.cruzi structure. Homologues of the C.jejuni dUTPase have been identified in several other bacteria and bacteriophages, including the dCTPase of phage T4. Sequence comparisons of these proteins define a new superfamily of d(C/U)TPases that includes three distinct enzyme families: (1) dUTPases in trypanosomatides, C.jejuni and several other Gram-negative bacteria, (2) predicted dUTPases in various Gram-positive bacteria and their phages, and (3) dCTP/dUTPases in enterobacterial T4-like phages. All these enzymes share a basic module that consists of two alpha-helices from the rigid domain, two helices from the mobile domain and connecting loops. These results in concert with a number of conserved residues responsible for interdomain cross-talk provide valuable insight towards rational drug design.

Amino Acid Sequence↗

PubMed2004

Purification, crystallization and preliminary X-ray diffraction analysis of the Trichoderma reesei hydrophobin HFBI.

Hydrophobins are fungal proteins that are capable of altering the hydrophobicity of surfaces by self-assembly at hydrophilic-hydrophobic interfaces. Here, the growth of hydrophobin crystals suitable for X-ray crystallography is reported. The hydrophobin HFBI from Trichoderma reesei was crystallized by vapour diffusion in hanging drops in 30% PEG 4000, 0.1 M sodium citrate pH 4.3 buffer containing 0.2 M ammonium acetate and CYMAL-5 detergent (initial concentration of 2.4 mM). HFBI crystals are hexagonal and belong to space group P6(1) (or P6(5)), with unit-cell parameters a = b = 45.9, c = 307.2 A. The HFBI used in the crystallization experiments was purified from fungal cell walls.

Crystallization↗

PubMed2004

Crystal structure reveals two alternative conformations in the active site of ribonuclease Sa2.

Three different strains of Streptomyces aureofaciens produce the homologous ribonucleases Sa, Sa2 and Sa3. The crystal structures of ribonuclease Sa (RNase Sa) and its complexes with mononucleotides have previously been reported at high resolution. Here, the structures of two crystal forms (I and II) of ribonuclease Sa2 (RNase Sa2) are presented at 1.8 and 1.5 A resolution. The structures were determined by molecular replacement using the coordinates of RNase Sa as a search model and were refined to R factors of 17.5 and 15.0% and R(free) factors of 21.8 and 17.2%, respectively. The asymmetric unit of crystal form I contains three enzyme molecules, two of which have similar structures to those seen for ribonuclease Sa, with Tyr87 at the bottom of their active sites. In the third molecule, Tyr87 has moved substantially: the CA atom moves almost 5 A and the OH of the side chain moves 10 A, inserting itself into the active site of a neighbouring molecule at a similar position to that observed for the nucleotide base in RNase Sa complexes. The asymmetric unit of crystal form II contains two Sa2 molecules, both of which are similar to the usual Sa structures. In one molecule, two main-chain conformations were modelled in the alpha-helix. Finally, a brief comparison is made between the conformations of the Sa2 molecules and those of 34 independent molecules taken from 20 structures of ribonuclease Sa and two independent molecules taken from two structures of ribonuclease Sa3 in various crystal forms.

Amino Acid Sequence↗

PubMed2004

Structure of a feruloyl esterase from Aspergillus niger.

The crystallographic structure of feruloyl esterase from Aspergillus niger has been determined to a resolution of 1.5 A by molecular replacement. The protein has an alpha/beta-hydrolase structure with a Ser-His-Asp catalytic triad; the overall fold of the protein is very similar to that of the fungal lipases. The structure of the enzyme-product complex was determined to a resolution of 1.08 A and reveals dual conformations for the serine and histidine residues at the active site.

Amino Acid Sequence↗

PubMed2004

Molecular details of urease inhibition by boric acid: insights into the catalytic mechanism.

The structure of the complex of urease, a Ni-containing metalloenzyme, with boric acid was determined at 2.10 A resolution. The complex shows the unprecedented binding mode of the competitive inhibitor to the dinuclear metal center, with the B(OH)3 molecule bridging the Ni ions and leaving in place the bridging hydroxide. Boric acid can be considered a substrate analogue of urea, and the structure supports the proposal that the Ni-bridging hydroxide acts as the nucleophile in the enzymatic process of urea hydrolysis.

Binding Sites↗

PubMed2004

The crystal structure of Trypanosoma cruzi dUTPase reveals a novel dUTP/dUDP binding fold.

dUTPase is an essential enzyme involved with nucleotide metabolism and replication. We report here the X-ray structure of Trypanosoma cruzi dUTPase in its native conformation and as a complex with dUDP. These reveal a novel protein fold that displays no structural similarities to previously described dUTPases. The molecular unit is a dimer with two active sites. Nucleotide binding promotes extensive structural rearrangements, secondary structure remodeling, and rigid body displacements of 20 A or more, which effectively bury the substrate within the enzyme core for the purpose of hydrolysis. The molecular complex is a trapped enzyme-substrate arrangement which clearly demonstrates structure-induced specificity and catalytic potential. This enzyme is a novel dUTPase and therefore a potential drug target in the treatment of Chagas' disease.

Amino Acid Sequence↗

PubMed2002

The structures of Micrococcus lysodeikticus catalase, its ferryl intermediate (compound II) and NADPH complex.

The crystal structure of the bacterial catalase from Micrococcus lysodeikticus has been refined using the gene-derived sequence both at 0.88 A resolution using data recorded at 110 K and at 1.5 A resolution with room-temperature data. The atomic resolution structure has been refined with individual anisotropic atomic thermal parameters. This has revealed the geometry of the haem and surrounding protein, including many of the H atoms, with unprecedented accuracy and has characterized functionally important hydrogen-bond interactions in the active site. The positions of the H atoms are consistent with the enzymatic mechanism previously suggested for beef liver catalase. The structure reveals that a 25 A long channel leading to the haem is filled by partially occupied water molecules, suggesting an inherent facile access to the active site. In addition, the structures of the ferryl intermediate of the catalase, the so-called compound II, at 1.96 A resolution and the catalase complex with NADPH at 1.83 A resolution have been determined. Comparison of compound II and the resting state of the enzyme shows that the binding of the O atom to the iron (bond length 1.87 A) is associated with increased haem bending and is accompanied by a distal movement of the iron and the side chain of the proximal tyrosine. Finally, the structure of the NADPH complex shows that the cofactor is bound to the molecule in an equivalent position to that found in beef liver catalase, but that only the adenine part of NADPH is visible in the present structure.

Amino Acid Sequence↗

PubMed2002

Atomic resolution data reveal flexibility in the structure of RNase Sa.

Ribonuclease from Streptomyces aureofaciens, the bacterial source for the industrial production of chlorotetracycline, is a guanylate endoribonuclease (RNase Sa; EC 3.1.27.3) which hydrolyses the phosphodiester bonds of single-stranded RNA at the 3'-side of guanosine nucleotides with high specificity. The structure of the enzyme was previously refined at atomic resolution (1.2 A) using room-temperature data. Here, the RNase Sa structure refined against 1.0 A data collected at cryogenic temperature is reported. There are two surface loops in molecule A and one in molecule B for which two main-chain conformations are modelled: these loops contain active-site residues. The separation for most of the corresponding main-chain atoms in the two conformations is about 0.8 A, with a maximum of 2.5 A. The two regions of dual conformation represent the most important differences in comparison with the structure determined at room temperature, where the corresponding loops have one conformation only but the largest degree of anisotropy. The flexibility of the loops observed in the structure of RNase Sa is directly linked to the need for the active site to interact productively with substrates and/or inhibitors.

Catalytic Domain↗

PubMed2002

The structure of Saccharomyces cerevisiae Met8p, a bifunctional dehydrogenase and ferrochelatase.

Sirohaem is a tetrapyrrole-derived prosthetic group that is required for the essential assimilation of sulfur and nitrogen into all living systems as part of the sulfite and nitrite reductase systems. The final two steps in the biosynthesis of sirohaem involve a beta-NAD(+)-dependent dehydrogenation of precorrin-2 to generate sirohydrochlorin followed by ferrochelation to yield sirohaem. In Saccharomyces cerevisiae, Met8p is a bifunctional enzyme that carries out both of these reactions. Here, we report the 2.2 A resolution crystal structure of Met8p, which adopts a novel fold that bears no resemblance to the previously determined structures of cobalt- or ferro-chelatases. Analysis of mutant proteins suggests that both catalytic activities share a single active site, and that Asp141 plays an essential role in both dehydrogenase and chelatase processes.

Amino Acid Sequence↗

PubMed2002

Domain closure, substrate specificity and catalysis of D-lactate dehydrogenase from Lactobacillus bulgaricus.

NAD-dependent Lactobacillus bulgaricus D-Lactate dehydrogenase (D-LDHb) catalyses the reversible conversion of pyruvate into D-lactate. Crystals of D-LDHb complexed with NADH were grown and X-ray data collected to 2.2 A. The structure of D-LDHb was solved by molecular replacement using the dimeric Lactobacillus helveticus D-LDH as a model and was refined to an R-factor of 20.7%. The two subunits of the enzyme display strong asymmetry due to different crystal environments. The opening angles of the two catalytic domains with respect to the core coenzyme binding domains differ by 16 degrees. Subunit A is in an "open" conformation typical for a dehydrogenase apo enzyme and subunit B is "closed". The NADH-binding site in subunit A is only 30% occupied, while in subunit B it is fully occupied and there is a sulphate ion in the substrate-binding pocket. A pyruvate molecule has been modelled in the active site and its orientation is in agreement with existing kinetic and structural data. On domain closure, a cluster of hydrophobic residues packs tightly around the methyl group of the modelled pyruvate molecule. At least three residues from this cluster govern the substrate specificity. Substrate binding itself contributes to the stabilisation of domain closure and activation of the enzyme. In pyruvate reduction, D-LDH can adapt another protonated residue, a lysine residue, to accomplish the role of the acid catalyst His296. Required lowering of the lysine pK(a) value is explained on the basis of the H296K mutant structure.

Binding Sites↗

PubMed2002

Low resolution solution structure of the Apo form of Escherichia coli haemoglobin protease Hbp.

We have studied the solution properties of the apo form of the haemoglobin protease or "haemoglobinase", Hbp, a principal component of an important iron acquisition system in pathogenic Escherichia coli. Experimental determination of secondary structure content from circular dichroism (CD) spectroscopy, obtained using synchrotron light, showed that the protein contains predominately beta-sheets in agreement with secondary structure prediction from the primary sequence. Next, the size and shape of the protein were probed using analytical ultracentrifugation (AUC) and small angle X-ray scattering (SAXS). These showed that Hbp is a monomer, with an extended conformation. Using ab initio reconstruction methods we have produced a model of Hbp, which shows that the protein adopts an extended crescent-shaped conformation. Analysis of the resulting model gives hydrodynamic parameters in good agreement with those observed experimentally. Thus we are able to construct a hydrodynamically rigorous model of apo-Hbp in solution, not only giving a greater level of confidence to the results of the SAXS reconstruction methods, but providing the first three-dimensional view of this intriguing molecule.

Apoenzymes↗