Structure, Function and Inhibition of Bacterial Oxidoreductases

Abstract

The disulfide bond forming proteins are a large and diverse family of proteins found in the periplasm of bacteria. They are involved in the maturation process, oxidation and isomerisation of disulfide bonds in their substrate periplasmic proteins, many of which are involved in pathogenicity and virulence pathways. Disulfide bond forming proteins are highly conserved yet do not appear to be critical for the survival of the bacteria under optimal conditions. Altogether these properties make them an ideal target to combat bacterial infections, with hopes and aims to reduce virulence of the bacteria while minimising selective pressure. This strategy is hypothesised to reduce the rate of resistance development compared to antibiotic and antimicrobial treatments. This thesis focuses specifically on the disulfide bond forming proteins A and B from Burkholderia pseudomallei, the bacterium causing the deadly tropical disease melioidosis. Chapter 2 focuses on screening a small natural products library using an assay reporting on the activity of the purified B. pseudomallei disulfide bond forming proteins A and B. Three natural products including two stilbenoids, (-)-hopeaphenol and vaticanol B, and an alkaloid, spermatinamine, were found to moderately inhibit the disulfide bond forming protein A and B redox reaction. Additionally, the stilbenoids were shown to bind to the disulfide bond forming protein A of B. pseudomallei. In chapter 3, X-ray crystallography was used to identify and characterise two druglike fragments from the Monash Institute for Pharmaceutical Sciences found to interact with B. pseudomallei disulfide bond forming protein A. These fragments were shown to bind a transient feature present only on the surface of the oxidised protein. In chapter 4, efforts focused on crystallising the complex of B. pseudomallei disulfide bond forming protein A with its membrane protein partner - disulfide bond forming protein B. Small weakly diffracting crystals of this complex were obtained using a technique for membrane protein crystallisation called lipidic cubic phase. Chapter 5 returns to B. pseudomallei disulfide bond forming protein A, with the objective to identify virulence-associated substrates. Having a better idea of what these substrates are, could for example, help identify antigenic epitopes for vaccine preparation or find reporters reliant on the activity of the disulfide bond forming proteins in phenotypic assays. In this research chapter, bioinformatic methods were used to filter and identify proteins suspected to be substrates of the B. pseudomallei disulfide bond forming protein A. Disulfide bond forming proteins have been extensively studied over the past 30 years, however they are not the only proteins involved in oxidoreduction reactions within the periplasm of bacteria. Other proteins, such as the suppressor of copper sensitivity proteins, have miscellaneous functions, including copper sequestration, disulfide bond oxidation and isomerisation. These proteins share structural similarities with the disulfide bond forming proteins, especially around the active site motif, however they differ in the way they oligomerise. Since only a few members of the suppressor of copper sensitivity protein family have been characterised to date, it is not entirely clear how their functions are dependent on their oligomerisation state. The last research chapter of this thesis, chapter 6, investigated the suppressor of copper sensitivity protein C from the organism Caulobacter crescentus. Biochemical characterisation indicated that this protein has a strong affinity for copper (I) and that it was also able to isomerise incorrectly formed disulfide bonds. Finally a crystal structure of the protein was obtained indicating trimerisation via a long α-helical Nterminal domain. In summary the work presented in this thesis investigated the structure, function and inhibition of bacterial oxidoreductases, specifically the disulfide bond forming proteins A and B from B. pseudomallei and the suppressor of copper sensitivity protein C from C. crescentus.