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Abstract: Room-temperature macromolecular crystallography allows protein structures to be determined under close-to-physiological conditions, permits dynamic freedom in protein motions and enables time-resolved studies. In the case of metalloenzymes that are highly sensitive to radiation damage, such room-temperature experiments can present challenges, including increased rates of X-ray reduction of metal centres and site-specific radiation-damage artefacts, as well as in devising appropriate sample-delivery and data-collection methods. It can also be problematic to compare structures measured using different crystal sizes and light sources. In this study, structures of a multifunctional globin, dehaloperoxidase B (DHP-B), obtained using several methods of room-temperature crystallographic structure determination are described and compared. Here, data were measured from large single crystals and multiple microcrystals using neutrons, X-ray free-electron laser pulses, monochromatic synchrotron radiation and polychromatic (Laue) radiation light sources. These approaches span a range of 18 orders of magnitude in measurement time per diffraction pattern and four orders of magnitude in crystal volume. The first room-temperature neutron structures of DHP-B are also presented, allowing the explicit identification of the hydrogen positions. The neutron data proved to be complementary to the serial femtosecond crystallography data, with both methods providing structures free of the effects of X-ray radiation damage when compared with standard cryo-crystallography. Comparison of these room-temperature methods demonstrated the large differences in sample requirements, data-collection time and the potential for radiation damage between them. With regard to the structure and function of DHP-B, despite the results being partly limited by differences in the underlying structures, new information was gained on the protonation states of active-site residues which may guide future studies of DHP-B.

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Abstract: A group-III iron containing 1,2-propanediol oxidoreductase, FucO, (also known as lactaldehyde reductase) from Escherichia coli was examined regarding its structure-dynamics-function relationships in the catalysis of the NADH dependent reduction of (2S)-lactaldehyde. Crystal structures of FucO variants in the presence or absence of cofactors have been determined, illustrating large domain movements between the apo and holo enzyme structures. Different structures of FucO variants co-crystallized with NAD+ or NADH together with substrate further suggest dynamic properties of the nicotinamide moiety of the coenzyme that are important for the reaction mechanism. Modeling of the native substrate (2S)-lactaldehyde into the active site can explain the stereoselectivity exhibited by the enzyme, with a critical hydrogen bond interaction between the (2S)-hydroxyl and the side-chain of N151, as well as the previously experimentally demonstrated pro-(R) selectivity in hydride transfer from NADH to the aldehydic carbon. Furthermore, the deuterium kinetic isotope effect of hydride transfer suggests that reduction chemistry is the main rate limiting step for turnover which is not the case in FucO catalyzed alcohol oxidation. We further propose that a water molecule in the active site – hydrogen bonded to a conserved histidine (H267) and the 2’-hydroxyl of the coenzyme ribose – functions as a catalytic proton donor in the protonation of the product alcohol. A hydrogen bond network of water molecules and the side chains of amino acid residues D360 and H267 links bulk solvent to this proposed catalytic water molecule.

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Abstract: A key step in the biosynthesis of numerous polyketides is the stereospecific formation of a spiroacetal (spiroketal). We report here that spiroacetal formation in the biosynthesis of the macrocyclic polyketides ossamycin and oligomycin involves catalysis by a novel spiroacetal cyclase. OssO from the ossamycin biosynthetic gene cluster (BGC) is homologous to OlmO, the product of an unannotated gene from the oligomycin BGC. The deletion of olmO abolished oligomycin production and led to the isolation of oligomycin-like metabolites lacking the spiroacetal structure. Purified OlmO catalyzed complete conversion of the major metabolite into oligomycin C. Crystal structures of OssO and OlmO reveal an unusual 10-strand β-barrel. Three conserved polar residues are clustered together in the β-barrel cavity, and site-specific mutation of any of these residues either abolished or substantially diminished OlmO activity, supporting a role for general acid/general base catalysis in spiroacetal formation.

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Abstract: The bacterial amino-acid transporter MhsT from the SLC6A family has been crystallized in complex with different substrates in order to understand the determinants of the substrate specificity of the transporter. Surprisingly, crystals of the different MhsT–substrate complexes showed interrelated but different crystal-packing arrangements. Space-group assignment and structure determination of these different crystal forms present challenging combinations of pseudosymmetry, twinning and translational non­crystallographic symmetry.

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Abstract: Silver (Ag(I)) displays multiple antimicrobial properties that have led to its widespread use in the medical field. However, extensive use of Ag(I) has led to the emergence of bacterial resistance to Ag(I). Resistance to Ag(I) was inferred through the presence of plasmid pMG101 that contains a gene cluster, sil, which allowed bacteria to survive six times the normal lethal dose of Ag(I). The proteins of the sil system were given putative functions based on their sequence homology to the more extensively studied cue and cus systems, involved in copper homeostasis. To date only SilE has been characterised. This work herein describes the functional and structural characterisation of three more of the proteins of the sil system; SilP, SilF and SilC. Functional characterisation involved the use of a variety of biophysical and biochemical assays, with the former giving information on the oligomeric state of the proteins and the effects of metal binding. The biochemical assays showed that the proteins are able to bind or interact with Ag(I) and Cu(I), with preferential binding to Ag(I). SilP, a P-type ATPase, activity assays suggest a modified catalytic cycle that challenges the current cycles attributed to other P-type ATPases. Structural studies utilised x-Ray crystallography to produce atomic models for both SilF and SilC. While SilP was investigated using Cryo-Electron Microscopy, which showed the protein is dimeric on grids and a viable target for future work. The functional and structural analysis within this thesis expands the limited understanding of the sil system and has significance for the future development of inhibitors of the proteins involved in bacterial silver resistance.