[show abstract][hide abstract] ABSTRACT: The paper reports the purification and characterization of the first penicillin acylase from Bacillus subtilis. YxeI, the protein annotated as hypothetical, coded by the gene yxeI in the open reading frame between iol and hut operons in B. subtilis was cloned and expressed in Eshcherichia coli, purified and characterized. The purified protein showed measurable penicillin acylase activity with penicillin V. The enzyme was a homotetramer of 148 kDa. The apparent K(m) of the enzyme for penicillin V and the synthetic substrate 2-nitro-5-(phenoxyacetamido)-benzoic acid was 40 mM and 0.63 mM, respectively, and the association constants were 8.93×10(2) M(-1) and 2.51×10(5) M(-1), respectively. It was inhibited by cephalosporins and conjugated bile salts, substrates of the closely related bile acid hydrolases. It had good sequence homology with other penicillin V acylases and conjugated bile acid hydrolases, members of the Ntn hydrolase family. The N-terminal nucleophile was a cysteine which is revealed by a simple removal of N-formyl-methionine. The activity of the protein was affected by high temperature, acidic pH and the presence of the denaturant guanidine hydrochloride.
International journal of biological macromolecules 01/2012; 50(1):25-30. · 2.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: Scientific contact lies at the heart of research and that between China and the U.K. is an important example of how it can come about. In 1911, when the Biochemical Society began, U.K. science was developing fast with profound discoveries in physics (the Rutherford atomic model) and biochemistry (the discovery of vitamins). In China, however, there was great social and political instability and a revolution. Since then, the turbulence of two world wars and a variety of deep global political tensions meant that the contacts between China and U.K. did not reflect the prodigious growth of biochemistry. There was, however, one particular and remarkable contact, that made by Joseph Needham, an outstanding biochemist. He visited China between 1943 and 1946, contacting many Chinese universities that were severely dislocated by war. Showing remarkable diplomatic abilities, Needham managed to arrange delivery of research and teaching equipment. His activities helped the universities to carry out their functions under near-impossible conditions and reminded them that they had friends abroad. Most remarkably, Joseph Needham developed an extraordinary grasp of Chinese culture, science and history and he opened the West to the extent and importance of Chinese science. Formal scientific and intellectual contacts between the scientific academic bodies in China and U.K., notably the Chinese Academy of Science and the Royal Society, resumed after British recognition of the Chinese Communist government in 1950. The delegations included outstanding scientists in biochemistry and related disciplines. Research activities, such as that concerning influenza, were soon established, whereas institutions, such as the Royal Society and the Wellcome Trust, acted a little later to support research. The outcomes have been long-term collaborations in such areas as insulin structure and function. There are now numerous joint activities in biochemistry and biomedicine supported by the MRC (Medical Research Council), BBSRC (Biotechnology and Biological Sciences Research Council), NERC (Natural Environment Research Council), EPSRC (Engineering and Physical Sciences Research Council) and UKRC (UK Research Councils). The present contacts and the associated research are very considerable and growing. It is clear that biochemistry in both countries has much to offer each other, and there is every reason to believe that these contacts will continue to expand in the future.
Biochemical Society Transactions 10/2011; 39(5):1313-22. · 2.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: Many critical cellular functions are performed by multisubunit circular protein oligomers whose internal geometry has evolved to meet functional requirements. The subunit number is arguably the most critical parameter of a circular protein assembly, affecting the internal and external diameters of the assembly and often impacting on the protein's function. Although accurate structural information has been obtained for several circular proteins, a lack of accurate information on alternative oligomeric states has prevented engineering such transitions. In this study we used the bacterial transcription regulator TRAP as a model system to investigate the features that define the oligomeric state of a circular protein and to question how the subunit number could be manipulated.
We find that while Bacillus subtilis and Bacillus stearothermophilus TRAP form 11-subunit oligomers, the Bacillus halodurans TRAP exclusively forms 12-subunit assemblies. Significantly, the two states of TRAP are related by a simple rigid body rotation of individual subunits around inter-subunit axes. We tested if such a rotation could be induced by insertion or deletion mutations at the subunit interface. Using wild type 11-subunit TRAP, we demonstrate that removal of five C-terminal residues at the outer side of the inter-subunit axis or extension of an amino acid side chain at the opposite, inner side, increased the subunit number from 11 to 12. Our findings are supported by crystal structures of TRAP oligomers and by native mass spectrometry data.
The subunit number of the TRAP oligomer can be manipulated by introducing deletion or addition mutations at the subunit interface. An analysis of available and emerging structural data on alternative oligomeric states indicates that the same principles may also apply to the subunit number of other circular assemblies suggesting that the deletion/addition approach could be used generally to engineer transitions between different oligomeric states.
PLoS ONE 01/2011; 6(10):e25296. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Insulin is a key protein hormone that regulates blood glucose levels and, thus, has widespread impact on lipid and protein metabolism. Insulin action is manifested through binding of its monomeric form to the Insulin Receptor (IR). At present, however, our knowledge about the structural behavior of insulin is based upon inactive, multimeric, and storage-like states. The active monomeric structure, when in complex with the receptor, must be different as the residues crucial for the interactions are buried within the multimeric forms. Although the exact nature of the insulin's induced-fit is unknown, there is strong evidence that the C-terminal part of the B-chain is a dynamic element in insulin activation and receptor binding. Here, we present the design and analysis of highly active (200-500%) insulin analogues that are truncated at residue 26 of the B-chain (B(26)). They show a structural convergence in the form of a new beta-turn at B(24)-B(26). We propose that the key element in insulin's transition, from an inactive to an active state, may be the formation of the beta-turn at B(24)-B(26) associated with a trans to cis isomerisation at the B(25)-B(26) peptide bond. Here, this turn is achieved with N-methylated L-amino acids adjacent to the trans to cis switch at the B(25)-B(26) peptide bond or by the insertion of certain D-amino acids at B(26). The resultant conformational changes unmask previously buried amino acids that are implicated in IR binding and provide structural details for new approaches in rational design of ligands effective in combating diabetes.
Proceedings of the National Academy of Sciences 02/2010; 107(5):1966-70. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Recent advances in computer hardware and software have led to the development of increasingly successful molecular simulations of protein structural dynamics that are intrinsic to biological processes. These simulations have resulted in models that increasingly agree with experimental observations, suggest new experiments and provide insights into biological mechanisms. Used in combination with data obtained with sophisticated experimental techniques, simulations are helping us to understand biological complexity at the atomic and molecular levels and are giving promising insights into the genetic, thermodynamic and functional/mechanistic behaviour of biological processes. Here, we highlight some examples of such approaches that illustrate the current state and potential of the field of molecular simulation.
[show abstract][hide abstract] ABSTRACT: Conformational transitions and functional stability of the bile salt hydrolase (BSH; cholylglycine EC: 188.8.131.52) from Bifidobacterium longum (BlBSH) cloned and expressed in E. coli were studied under thermal, chemical and pH-mediated denaturation conditions using fluorescence and CD spectroscopy. Thermal and Gdn-HCl-mediated denaturation of BlBSH is a multistep process of inactivation and unfolding. The inactivation and unfolding of the enzyme was found to be irreversible. Enzyme activity seems sensitive to even minor conformational changes at the active site. Thermal denaturation as such did not result in any insoluble protein aggregates. However, on treating with 0.25 - 1 M Gdn-HCl the enzyme showed increasing aggregation at temperatures of 40 - 55 degrees C indicating more complex structural changes taking place in the presence of chemical denaturants. The enzyme secondary structure was still intact at acidic pH (pH 1 - 3). The perturbation in the tertiary structure at the acidic pH was detected through freshly formed solvent exposed hydrophobic patches on the enzyme. These changes could be due to the formation of an acid-induced molten globule-like state.
International Union of Biochemistry and Molecular Biology Life 03/2007; 59(2):118-25. · 2.79 Impact Factor
[show abstract][hide abstract] ABSTRACT: Bile salt hydrolase (BSH) is an enzyme produced by the intestinal microflora that catalyzes the deconjugation of glycine- or taurine-linked bile salts. The crystal structure of BSH reported here from Bifidobacterium longum reveals that it is a member of N-terminal nucleophil hydrolase structural superfamily possessing the characteristic alphabetabetaalpha tetra-lamellar tertiary structure arrangement. Site-directed mutagenesis of the catalytic nucleophil residue, however, shows that it has no role in zymogen processing into its corresponding active form. Substrate specificity was studied using Michaelis-Menten and inhibition kinetics and fluorescence spectroscopy. These data were compared with the specificity profile of BSH from Clostridium perfrigens and pencillin V acylase from Bacillus sphaericus, for both of which the three-dimensional structures are available. Comparative analysis shows a gradation in activity toward common substrates, throwing light on a possible common route toward the evolution of pencillin V acylase and BSH.
Journal of Biological Chemistry 11/2006; 281(43):32516-25. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The nature of the factors leading to the conversion of the cellular prion protein (PrP(C)) into its amyloidogenic isoform (PrP(Sc)) is still matter of debate in the field of structural biology. The NMR structures of non-mammalian PrP(C) (non-mPrP) from frog, chicken and turtle [Calzolai, L., Lysek, D.A., Perez, D.R., Guntert, P. and Wuthrich, K. (2005) Prion protein NMR structures of chickens, turtles, and frogs. Proc. Natl. Acad. Sci. USA 102, 651-655] have provided some new and valuable information on the scaffolding elements that preserve the PrP(C) folding, despite their low sequence identity with the mammalian prions (mPrP). The present molecular dynamics study of non-mPrP(C) focuses on the hydration properties of these proteins in comparison with the mammalian ones. The data reveal new insights in the PrP hydration and focus on the implications for PrP(C) folding stability and its propensity for interactions. In addition, for the first time, a role in disfavoring the PrP(C) aggregation is suggested for a conserved beta-bulge which is stabilized by the local hydration.
[show abstract][hide abstract] ABSTRACT: Penicillin acylase proteins are amidohydrolase enzymes that cleave penicillins at the amide bond connecting the side chain to their beta-lactam nucleus. An unannotated protein from Bacillus subtilis has been expressed in Escherichia coli, purified and confirmed to possess penicillin V acylase activity. The protein was crystallized using the hanging-drop vapour-diffusion method from a solution containing 4 M sodium formate in 100 mM Tris-HCl buffer pH 8.2. Diffraction data were collected under cryogenic conditions to a spacing of 2.5 A. The crystals belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 111.0, b = 308.0, c = 56.0 A. The estimated Matthews coefficient was 3.23 A3 Da(-1), corresponding to 62% solvent content. The structure has been solved using molecular-replacement methods with B. sphaericus penicillin V acylase (PDB code 2pva) as the search model.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 08/2005; 61(Pt 7):680-3. · 0.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: The propensity to form fibril in disease-related proteins is a widely studied phenomenon, but its correlation, if any, with structural characteristics of the associated proteins is not clearly understood. However, the observation has been made that some proteins that readily form amyloid have a significant number of backbone H bonds that are exposed to solvent molecules, suggesting that these regions have a propensity toward protein interaction and aggregation [Fernandez, A. & Scheraga, H. A. (2003) Proc. Natl. Acad. Sci. USA 100, 113-118]. High-resolution x-ray structures of the sheep and human C-terminal prion protein have provided a useful description of surface and partially buried waters. By molecular dynamics simulations, we investigated the structural role of these water molecules. The solvent dynamical behavior on the protein surface reveals significant features about the stability and the potential interactions of the prion protein. The protein presents regions of tightly bound conserved waters that are necessary to hold in place local elements of the fold, as well as regions where the local water is in fast exchange with bulk water. These results are evidenced by a map of the spatial distribution entropy of the solvent around the protein. The particular behavior of the solvent around these regions may be crucial in the folding stability and in terms of aggregation loci.
Proceedings of the National Academy of Sciences 06/2005; 102(21):7535-40. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Conjugated bile salt hydrolase (BSH) catalyses the hydrolysis of the amide bond that conjugates bile acids to glycine and to taurine. The BSH enzyme from Bifidobacterium longum was overexpressed in Escherichia coli BL21(DE3), purified and crystallized. Crystallization conditions were screened using the hanging-drop vapour-diffusion method. Crystal growth, with two distinct morphologies, was optimal in experiments carried out at 303 K. The crystals belong to the hexagonal system, space group P622 with unit-cell parameters a = b = 124.86, c = 219.03 A, and the trigonal space group P321, with unit-cell parameters a = b = 125.24, c = 117.03 A. The crystals diffracted X-rays to 2.5 A spacing. Structure determination using the multiple isomorphous replacement method is in progress.
[show abstract][hide abstract] ABSTRACT: An experimentally determined pK(a) change of +2.50 units has been reported for the B13 Glu residue in a dimeric B9 Ser --> Asp insulin mutant relative to the native dimer. Poisson-Boltzmann electrostatics-based pK(a) calculations were performed to probe the effect of the B9 Ser --> Asp and B10 His --> Asp mutations on aggregation and the ionization behaviour of the B13 carboxylate. The method produced shifts of +2.64 and +2.45 units for the pK(a) shift of the two B13 residues in the B9 mutant dimer relative to the wild-type dimer, which is in good agreement with the experimental value (<6% error). The calculations also suggest that the reason neither mutant insulin can aggregate into hexamers is the resultant crowding of negatively charged groups in the central solvent channel on hexamer formation. In the wild-type insulin, binding of zinc ions by B10 His overcomes this problem, whereas in the B10 mutant this possibility is ruled out by the absence of the zinc binding site. A series of mutations are predicted to stabilize the medically relevant, monomeric form of insulin.
Protein Engineering Design and Selection 08/2004; 17(7):557-63. · 2.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: Both prion protein and the structurally homologous protein doppel are associated with neurodegenerative disease by mechanisms which remain elusive. We have prepared murine doppel, and a mutant with one of the two disulphide bonds removed, in the expectation of increasing the similarity of doppel to prion protein in terms of conformation and stability. Unfolding studies of doppel and the mutant have been performed using far-UV CD over a range of solution conditions known to favour the alpha-->beta transformation of recombinant prion protein. Only partial unfolding of doppel or the mutant occurs at elevated temperature, but both exhibit full and reversible unfolding in chemical denaturation with urea. Doppel is significantly less stable than prion protein, and this stability is further reduced by removal of the disulphide bond between residues 95-148. Both doppel and the mutant are observed to unfold by a two-state mechanism, even under the mildly acidic conditions where prion protein forms an equilibrium intermediate with enhanced beta-structure, potentially analogous to the conversion of the cellular form of the prion protein into the infectious form (PrP(C)-->PrP(Sc)). Furthermore, no direct interaction of either doppel protein with prion protein, either in the alpha-form or the beta-rich conformation, was detectable spectroscopically. These studies indicate that, in spite of the similarity in secondary structure between the doppel and prion protein, there are significant differences in their solution properties. The fact that neither doppel nor its mutant exhibited the alpha-->beta transformation of the prion protein suggests that this conversion property may be dependent on unique sequences specific to the prion protein.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Penicillin acylases are used in the pharmaceutical industry for the preparation of antibiotics. The 3-D structure of Penicillin
Gacylase from Escherichia coli has been solved. Here, we present structural data that pertain to the unanswered questions that arose from the original strucutre.
Specificity for the amide portion of substrate was probed by the structure determination of a range of complexes with substitutions
around the phenylacetyl ring of the ligand. Altered substrate specificity mutations derived from an in vivo positive selection
process have also been studied, revealing the structural consequences of mutation at position B71.
Protein processing has been analyzed by the construction of site-directed mutants, which affect this reaction with two distinct
phenotypes. Mutations that allow processing but yield inactive protein provide the structure of an ES complex with a true
substrate, with implications for the enzymatic mechanism and stereospecificity of the reaction. Mutations that preclude processing
have allowed the structure of the precursor, which includes the 54a mino acid linker region normally removed from between
the A and B chains, to be visualized.
Applied Biochemistry and Biotechnology 06/2000; 88(1):313-319. · 1.89 Impact Factor