Structural Characterisation of the Beta-Ketoacyl-Acyl Carrier Protein Synthases, FabF and FabH, of Yersinia pestis.

Yersinia pestis, the causative agent of bubonic, pneumonic, and septicaemic plague, remains a major public health threat, with outbreaks of disease occurring in China, Madagascar, and Peru in the last five years. The existence of multidrug resistant Y. pestis and the potential of this bacterium as a bioterrorism agent illustrates the need for new antimicrobials. The ß-ketoacyl-acyl carrier protein synthases, FabB, FabF, and FabH, catalyse the elongation of fatty acids as part of the type II fatty acid biosynthesis (FASII) system, to synthesise components of lipoproteins, phospholipids, and lipopolysaccharides essential for bacterial growth and survival. As such, these enzymes are promising targets for the development of novel therapeutic agents. We have determined the crystal structures of the Y. pestis ß-ketoacyl-acyl carrier protein synthases FabF and FabH, and compared these with the unpublished, deposited structure of Y. pestis FabB. Comparison of FabB, FabF, and FabH provides insights into the substrate specificities of these enzymes, and investigation of possible interactions with known ß-ketoacyl-acyl carrier protein synthase inhibitors suggests FabB, FabF and FabH may be targeted simultaneously to prevent synthesis of the fatty acids necessary for growth and survival.

An efficient approach for recombinant expression and purification of the viral capsid protein from beak and feather disease virus (BFDV) in Escherichia coli.

Structural insights into the biology of viruses such as beak and feather disease virus (BFDV) which do not replicate in cell cultures are increasingly reliant on recombinant methods for protein production and purification. Development of efficient methods for homogenous production of BFDV capsid protein is also essential for vaccine development and diagnostic purposes. In this study, two different plasmids (pMCSG21 and pMCSG24), three homologous BFDV capsid proteins, and two unique expression media (auto-induction and IPTG-induced expression) were trialled for over-expression of the BFDV in Escherichia coli. Over-expression was observed for all three recombinant targets of BFDV capsid protein using E. coli BL21 (DE3) Rosetta 2 cell lines under IPTG induction. These proteins could be purified using an optimized, two-step purification process using a buffer containing 20mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), 500 mM NaCl and supplemented with 200 mM L-arginine at pH 10.5, to yield a soluble and stable protein of greater than 95% purity. The final concentration of purified protein was approximately fourteen-to-eighteen fold greater than that reported previously. Initial crystallization and X-ray diffraction confirm that the protein is structured in a manner consistent with icosahedral symmetry. Antigenicity of recombinant Cap was confirmed by immunoassay, verifying its validity for use in continued experimentation as a potential DNA vaccine, a reagent in diagnostic assays, and purified concentrated protein for structural and functional biology.

Structural characterization of a Gcn5-related N-acetyltransferase from Staphylococcus aureus.

The Gcn5-related N-acetyltransferases (GNATs) are ubiquitously expressed in nature and perform a diverse range of cellular functions through the acetylation of small molecules and protein substrates. Using activated acetyl coenzyme A as a common acetyl donor, GNATs catalyse the transfer of an acetyl group to acceptor molecules including aminoglycoside antibiotics, glucosamine-6-phosphate, histones, serotonin and spermidine. There is often only very limited sequence conservation between members of the GNAT superfamily, in part, reflecting their capacity to bind a diverse array of substrates. In contrast, the secondary and tertiary structures are highly conserved, but then at the quaternary level there is further diversity, with GNATs shown to exist in monomeric, dimeric, or tetrameric states. Here we describe the X-ray crystallographic structure of a GNAT enzyme from Staphylococcus aureus with only low sequence identity to previously solved GNAT proteins. It contains many of the classical GNAT motifs, but lacks other hallmarks of the GNAT fold including the classic ß-bulge splayed at the ß-sheet interface. The protein is likely to be a dimer in solution based on analysis of the asymmetric unit within the crystal structure, homology with related GNAT family members, and size exclusion chromatography. The study provides the first high resolution structure of this enzyme, providing a strong platform for substrate and cofactor modelling, and structural/functional comparisons within this diverse enzyme superfamily.