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SSPF HighlightsThe following is a list of highlights from the facility. Click on each for further information and pdb entry details1. Sulfolobus Solfataricus RadA- RadA is a striking example of an enzyme which is far more similar to its human counterpart (Rad51) than any bacterial equivalent (e.g. RecA). The crystal structure of S. solfataricus RadA has informed us about how flexibility in this filament-forming protein is controlled. 2. Tryptophan 7-halogenase (PrnA)- Chlorinated natural products include vancomycin and cryptophycin A. Their biosyntheses involves regioselective chlorination by flavin-dependent halogenases. Tryptophan 7-halogenase (PrnA), which regioselectively chlorinates tryptophan. Tryptophan and FAD are separated by a 10A-long tunnel and bound by distinct enzyme modules. The FAD module is conserved in halogenases and is related to flavin-dependent monooxygenases. Based on biochemical studies, crystal structures and by analogy with monooxygenases, we predict FADH2 reacts with O2 making peroxy-flavin which is decomposed by Cl-. The resulting HOCl is guided through the tunnel, to tryptophan, where it is activated. 3. Alba2- The Alba proteins are important DNA binding proteins in archaea, involved in DNA packaging. The Alba1 protein is modulated by reversible acetylation of a lysine residue, reminiscent of eukaryal histones. We demonstrate that Alba1 and Alba2 form obligate heterodimers, and have solved the crystal structure of the heterodimer. NMR, an electron microscopy studies suggest that this dimerisation may play an important role in DNA packaging, by altering packing of successive dimers during assembly into a chromatin structure. 4. Sialidase- Sialidases hydrolyse the removal of sialic acid from a variety of glycoconjugates, and are retaining enzymes. Through mutation of the nucleophilic tyrosine to a glycine, we have engineered a sialidase that acts through inversion by allowing a water molecule to attack from below. Depending on the leaving group, the inverting sialidase is as efficient as the retaining enzyme, and we are exploring its use in biotransformations. 5. Streptomyces coelicolor- Analyses of microbial genome sequences reveal numerous examples of gene clusters encoding proteins typically involved in complex natural product biosynthesis but not associated with the production of known natural products. We report the isolation and structure determination of the new tris-hydroxamate tetrapeptide iron chelator coelichelin from S. coelicolor using a genome mining approach guided by substrate predictions for the trimodular NRPS CchH. These results demonstrate that accurate prediction of adenylation domain substrate selectivity is possible and raise intriguing mechanistic questions regarding the assembly of a tetrapeptide by a trimodular NRPS. 6. Hydroxypropylphosphonic acid epoxidase- Fosfomycin is a clinically useful antibiotic with an unusual oxirane structure synthesised by a unique epoxidase. The structure and biochemical data suggest a novel mechanism and the resulting model may allow for the development of altered enzymes able to synthesise new antibiotics. 7. Serine Palmitoyl transferase- Serine Palmitoyl transferase catalyses the first step in Sphingolipid synthesis, the condensation of serine and palmitoyl CoA. This enzyme is mutated in the human disease Hereditary sensory neuropathy type 1. 8. Putative Phosphofructokinase- This protein from Staphylococcus aureus is a putative phosphofructokinase, which converts fructose 1-phosphate to fructose 1,6-bisphosphate and is part of the fructose metabolism. 9. Wza- Gram-negative bacteria need to be able to transport a large variety of macromolecules across their outer membranes. In Escherichia coli, the passage of the group 1 capsular polysaccharide is mediated by an integral outer membrane protein, Wza. The crystal structure of Wza reveals a novel transmembrane alpha-helical barrel and a large central cavity within the core of the vase-shaped protein complex. 10. Sso1404- Sso1404 is part of COG1343, which contains the Cas2(CRISPR Associated 2) family of proteins. CRISPR's (Clustered regularly Interspaced Short Palindromic Repeats) are short regions of palindromic DNA identified in bacteria and archea, which have been found to be homologous to fragments of phage and plasmid genes. It is hypothesized that CASPRs are involved in a RNA interference (RNAi) defence mechanism. 11. 3-Methyladenine DNA Glycosylase I- This protein is part of the DNA Excision-repair pathway in bacteria. This is the first crystal structure of this enzyme. 12. PCNA- PCNA is a ring-shaped protein that encircles DNA, providing a platform for the association of a wide variety of DNA-processing enzymes that utilize the PCNA sliding clamp to maintain proximity to their DNA substrates 13. Acsd- AcsD from in Pectobacterium chrysanthemi belongs to type A siderophores synthetase in the NRPS-independent Siderophore Biosynthetic pathway (Challis, ChemBioChem 2005, 6, 601 – 611). It is involved in the synthesis of Achromobactin. The structure shows the monomer with one bound compound citrate in the active site. 14.
15. Ranasmurfin- Ranasmurfin, a previously uncharacterized approximately 13 kDa blue protein found in the nests of the frog Polypedates leucomystax, has been purified and crystallized. Sequence data for the protein are incomplete, but so far there has been no identified model for molecular replacement. A fluorescence scan shows a peak at 9.676 keV, indicating that the protein binds zinc and suggesting a route for structure solution |
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