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A M Deacon

Publications and source records attributed to A M Deacon.

6 recordsLinked to original sources
PubMed2000

The crystal structure of ADP-L-glycero-D-mannoheptose 6-epimerase: catalysis with a twist.

BACKGROUND: ADP-L-glycero--mannoheptose 6-epimerase (AGME) is required for lipopolysaccharide (LPS) biosynthesis in most genera of pathogenic and non-pathogenic Gram-negative bacteria. It catalyzes the interconversion of ADP-D-glycero-D-mannoheptose and ADP-L-glycero-D-mannoheptose, a precursor of the seven-carbon sugar L-glycero-mannoheptose (heptose). Heptose is an obligatory component of the LPS core domain; its absence results in a truncated LPS structure resulting in susceptibility to hydrophobic antibiotics. Heptose is not found in mammalian cells, thus its biosynthetic pathway in bacteria presents a unique target for the design of novel antimicrobial agents. RESULTS: The structure of AGME, in complex with NADP and the catalytic inhibitor ADP-glucose, has been determined at 2.0 A resolution by multiwavelength anomalous diffraction (MAD) phasing methods. AGME is a homopentameric enzyme, which crystallizes with two pentamers in the asymmetric unit. The location of 70 crystallographically independent selenium sites was a key step in the structure determination process. Each monomer comprises two domains: a large N-terminal domain, consisting of a modified seven-stranded Rossmann fold that is associated with NADP binding; and a smaller alpha/beta C-terminal domain involved in substrate binding. CONCLUSIONS: The first structure of an LPS core biosynthetic enzyme leads to an understanding of the mechanism of the conversion between ADP-D-glycero--mannoheptose and ADP-L-glycero-D-mannoheptose. On the basis of its high structural similarity to UDP-galactose epimerase and the three-dimensional positions of the conserved residues Ser116, Tyr140 and Lys144, AGME was classified as a member of the short-chain dehydrogenase/reductase (SDR) superfamily. This study should prove useful in the design of mechanistic and structure-based inhibitors of the AGME catalyzed reaction.

Adenosine Diphosphate Glucose↗
PubMed2000

Ill-conditioned Shake-and-Bake: the trap of the false minimum.

The alternation of phase refinement with the imposition of real-space constraints is the essence of the Shake-and-Bake procedure. Typically, these constraints prevent trial structures from falling into local minima. Nevertheless, P1 structures appear to migrate to false minima with significant frequency. These false minima are characterized by the presence of a large 'uranium' peak on the corresponding Fourier map. Fortunately, they can be recognized and avoided by considering the values of the minimal function both before and after the application of constraints. However, it appears that finding solutions for large P1 structures is likely also to require parameter-shift conditions different from those that have been found to work well in other space groups. In fact, these conditions often yield an unusually high percentage of solutions.

Algorithms↗
PubMed2000

The atomic resolution structure of bucandin, a novel toxin isolated from the Malayan krait, determined by direct methods.

Bucandin is a novel presynaptic neurotoxin isolated from Bungarus candidus (Malayan krait). It has the unique property of enhancing presynaptic acetylcholine release and represents a family of three-finger toxins with an additional disulfide in the first loop. There are no existing structures from this sub-category of three-finger toxins. The X-ray crystal structure of bucandin has been determined by the Shake-and-Bake direct-methods procedure. The resulting electron-density maps were of outstanding quality and allowed the automated tracing of 61 of the 63 amino-acid residues, including their side chains, and the placement of 48 solvent molecules. The 0.97 A resolution full-matrix least-squares refinement converged to a crystallographic R factor of 12.4% and the final model contains 118 solvent molecules. This is the highest resolution structure of any member of the three-finger toxin family and thus it can serve as the best model for other members of the family. Furthermore, the structure of this novel toxin will help in understanding its unique ability to enhance acetylcholine release. The unique structure resulting from the fifth disulfide bond residing in the first loop improves the understanding of other toxins with a similar arrangement of disulfide bonds.

Amino Acid Sequence↗
PubMed1999

Crystallization and preliminary X-ray diffraction studies of the lipopolysaccharide core biosynthetic enzyme ADP-L-glycero-D-mannoheptose 6-epimerase from Escherichia coli K-12.

ADP-L-glycero-D-mannoheptose 6-epimerase is a 240 kDa NAD-dependent nucleotide diphosphosugar epimerase from Escherichia coli K12 which catalyzes the interconversion of ADP-D-glycero-D-mannoheptose and ADP-L-glycero-D-mannoheptose. ADP-L-glycero-D-mannoheptose is a required intermediate for lipopolysaccharide inner-core and outer-membrane biosynthesis in several genera of pathogenic and non-pathogenic Gram-negative bacteria. ADP-L-glycero-D-mannoheptose 6-epimerase was overexpressed in E. coli and purified to apparent homogeneity by chromatographic methods. Three crystal forms of the epimerase were obtained by a hanging-drop vapor-diffusion method. A native data set for crystal form III was collected in-house on a Rigaku R-AXIS-IIC image plate at 3.0 A resolution. The form III crystals belong to the monoclinic space group P21. The unit-cell parameters are a = 98.94, b = 110.53, c = 180.68 A and beta = 90.94 degrees. Our recent results show that these crystals diffract to 2.0 A resolution at the Cornell High Energy Synchrotron Source. The crystal probably contains six 40 kDa monomers per asymmetric unit, with a corresponding volume per protein mass (Vm) of 4.11 A3 Da-1 and a solvent fraction of 70%.

Carbohydrate Epimerases↗
PubMed1998

The Shake-and-Bake structure determination of triclinic lysozyme.

The crystal structure of triclinic lysozyme, comprised of 1,001 non-H protein atoms and approximately 200 bound water molecules, has been determined ab initio (using native data alone) by the "Shake-and-Bake" method by using the computer program SnB. This is the largest structure determined so far by the SnB program. Initial experiments, using default SnB parameters derived from studies of smaller molecules, were unsuccessful. In fact, such experiments produced electron density maps dominated by a single large peak. This problem was overcome by considering the choice of protocol used during the parameter-shift phase refinement. When each phase was subjected to a single shift of +/-157.5 degrees during each SnB cycle, an unusually high percentage of random trials (approximately 22%) yielded correct solutions within 750 cycles. This success rate is higher than that typically observed, even for much smaller structures.

Crystallography, X-Ray↗