The myocilin olfactomedin domain (myoc-OLF) is linked to inherited forms of open angle glaucoma. Mutant myocilin accumulates within the endoplasmic reticulum of human trabecular meshwork cells leading to cell death and the build up of intraocular pressure, a common risk factor for glaucoma. In this work, a novel high affinity calcium-binding site buried within myoc-OLF was characterized. Additionally amyloidogenic peptide stretches within myoc-OLF that may be responsible for mutant myocilin aggregation were determined. Additionally the crystal structure of myoc-OLF was solved providing the first crystal structure of an olfactomedin domain protein. Insights from the structure into the relationship between disease causing mutations and myoc-OLF misfolding and the currently unknown function of myoc-OLF as explored by structural based prediction of ligand binding are also discussed.

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An enzyme has been isolated from the fungus responsible for Dutch Elm Disease, Ophiostoma novo-ulmi, that stereoselectively catalyses the production of S ketoprofen from racemic ethyl ketoprofen. The enzyme has been cloned, over-expressed and mutated by Chiroscience, plc. The main aim, at the outset of this work, was to crystallise this enzyme and determine the three-dimensional crystallographic structure. A mutant form of the enzyme was purified to apparent homogeneity, by two different protocols, and subjected to numerous crystallisation trials. Trials were unsuccessful. Mass spectrometry of the purified sample revealed the presence of two protein species that differed by 832 Da. Analysis of the expression vector and N-terminal sequences of the two protein species revealed that they were both forms of the enzyme that differed by ten amino acids at the N-terminus. A further sample was supplied that contained only one form of the enzyme. This has been purified by the same two purification protocols and subjected to similar crystallisation trials. Needle like crystals have been grown. In order to collect X-ray diffraction data it has been necessary to carry out extensive crystallisation trials with the enzyme. In this respect the work developed into a study of crystallisation techniques and phenomena, which focused on uncoupling the processes of nucleation and growth and generally controlling nucleation. Crystals have been produced that diffract beyond 2.5 A and a native dataset has been collected at the Daresbury synchrotron source. Heavy atom derivatives have so far been nonisomorphous. An immobilised form of the enzyme has been produced by cross linking microcrystals with the bifunctional reagent glutaraldehyde. In this form the enzyme has increased stability towards temperature, pH and organic solvents. Amino acid sequence alignment and chemical analyses have suggested that the enzyme may belong to a superfamily of active serine hydrolases that include penicillin recognising proteins.The vanadium dependent bromoperoxidase from the macroalgae Corallina officinalis has been purified to homogeneity and crystallised. Native and derivative X-ray diffraction datasets have been collected and the structure solved by Multiple Isomorphous Replacement in collaboration with Dr M. Isupov and Dr A. Dalby. The molecule displays 23 point symmetry, which has not been observed in proteins to date. The molecule was crystallised in its dodecameric form, revealing the monomer to be a single domain a-helical protein with a four helix bundle as the main structural motif. The vanadium binding site is located at the end of this four helix bundle. The vanadium binding site of the chloroperoxidase from Cul. inaequalis is also located at the end of a four helix bundle. This illustrates that although there is limited amino acid sequence homology between the two enzymes they are structurally related. Purification

Inducible chomosomal AmpC β-lactamase (AmpC) is present in many Gram-negative opportunistic human pathogens. Expressed in response to β-lactam antibiotics, AmpC is an enzyme that can deactivate an extended spectrum of β-lactam antibiotics and thereby promote bacterial survival. Inducible chromosomal ampC is associated with ampR, a gene that encodes a LysR-type transcriptional regulator that suppresses ampC expression in the absence of β-lactam exposure. Together, ampR and ampC form a divergent operon with overlapping promoters to which the AmpR protein binds and regulates the transcription of both genes. AmpR induces ampC expression by interacting with 1,6-anhydro-N-acetylmuramyl peptide, an intermediate of peptidoglycan recycling that is generated by a glycoside hydrolase encoded by nagZ. Given the role of NagZ and AmpR in the AmpC induction pathway, the structure and function of these proteins were investigated to understand the molecular basis for how they participate in AmpC production. The crystal structure of NagZ from Vibrio cholerae was determined in complex with the glycoside hydrolase inhibitor PUGNAc (O-(2-Deoxy-2-N-2-ethylbutyryl-D-glucopyranosylidene)amino-N-phenylcarbamate) to 1.8 Å resolution. Since PUGNAc also inhibits functionally related human enzymes, the structure of the enzyme was also determined in complex with the NagZ selective PUGNAc derivatives N-butyryl-PUGNAc (2.3 Å resolution) and N-valeryl-PUGNAc (2.4 Å resolution). These structural studies revealed the molecular basis for how 2-N-acyl derivatives of PUGNAc selectively inhibit the bacterial enzyme NagZ. The effector binding domain of AmpR from Citrobacter Spp. was determined to 1.83 Å resolution and lead to the identification of a putative effector molecule binding site. In vivo functional analysis of site directed mutants of AmpR containing amino acid substitutions at the base of the putative binding pocket verified its role in AmpR function. A protocol was subsequently devised to purify milligram quantities of soluble full-length AmpR. Biochemical and biophysical analysis, including non-denaturing mass spectrometry and small angle X-ray scattering, revealed that the purified full-length protein is tetrameric and specifically binds ampC promoter DNA. In summary, this research provides the basis for the development of small-molecules that could specifically block the activity of these proteins to suppress AmpC β-lactamase production during β-lactam therapy.

Inducible chomosomal AmpC β-lactamase (AmpC) is present in many Gram-negative opportunistic human pathogens. Expressed in response to β-lactam antibiotics, AmpC is an enzyme that can deactivate an extended spectrum of β-lactam antibiotics and thereby promote bacterial survival. Inducible chromosomal ampC is associated with ampR, a gene that encodes a LysR-type transcriptional regulator that suppresses ampC expression in the absence of β-lactam exposure. Together, ampR and ampC form a divergent operon with overlapping promoters to which the AmpR protein binds and regulates the transcription of both genes. AmpR induces ampC expression by interacting with 1,6-anhydro-N-acetylmuramyl peptide, an intermediate of peptidoglycan recycling that is generated by a glycoside hydrolase encoded by nagZ. Given the role of NagZ and AmpR in the AmpC induction pathway, the structure and function of these proteins were investigated to understand the molecular basis for how they participate in AmpC production. The crystal structure of NagZ from Vibrio cholerae was determined in complex with the glycoside hydrolase inhibitor PUGNAc (O-(2-Deoxy-2-N-2-ethylbutyryl-D-glucopyranosylidene)amino-N-phenylcarbamate) to 1.8 Å resolution. Since PUGNAc also inhibits functionally related human enzymes, the structure of the enzyme was also determined in complex with the NagZ selective PUGNAc derivatives N-butyryl-PUGNAc (2.3 Å resolution) and N-valeryl-PUGNAc (2.4 Å resolution). These structural studies revealed the molecular basis for how 2-N-acyl derivatives of PUGNAc selectively inhibit the bacterial enzyme NagZ. The effector binding domain of AmpR from Citrobacter Spp. was determined to 1.83 Å resolution and lead to the identification of a putative effector molecule binding site. In vivo functional analysis of site directed mutants of AmpR containing amino acid substitutions at the base of the putative binding pocket verified its role in AmpR function. A protocol was subsequently devised to purify milligram quantities of soluble full-length AmpR. Biochemical and biophysical analysis, including non-denaturing mass spectrometry and small angle X-ray scattering, revealed that the purified full-length protein is tetrameric and specifically binds ampC promoter DNA. In summary, this research provides the basis for the development of small-molecules that could specifically block the activity of these proteins to suppress AmpC β-lactamase production during β-lactam therapy.