Investigation of the pyridoxine/pyridoxamine 5’-phosphate oxidase family of enzymes in mycobacteria

Abstract

Tuberculosis (TB) is a deadly disease that is believed to infect about one-third of the world’s population. It is caused by Mycobacterium tuberculosis (Mtb), a slow growing, Gram-positive bacterium that has evolved to adapt and survive in human host. Although predominantly an aerobic bacterium, Mtb is known to survive under hypoxic conditions inside host macrophages in a state of non-replicating persistence (NRP), which can be attributed to switches in its metabolic processes to oxygen-independent mechanisms. Recent developments in research have shown that cofactor F420, a 5-deazaflavin derivative, plays a vital role in Mtb metabolism. F420 has been implicated in the redox mechanisms that protect the bacterium from oxidative and nitrosative stress conditions inside host cells. Furthermore, recent bioinformatics studies have suggested the presence of an unexpected number of F420-dependent enzymes in Mtb. The aim of this project was to investigate the role of F420 in the metabolic adaptations of Mtb through functional and structural studies of the classical FMN-dependent and the annotated F420-dependent pyridoxine/pyridoxamine 5’-phosphate oxidase (PNPOx) family of enzymes. The classical FMN-dependent PNPOx (PdxH) enzymes from four mycobacterial species were expressed as soluble proteins in M. smegmatis host cells. The classical enzymes were purified as bright yellow-coloured proteins, containing FMN as a prosthetic group. In addition, activity assays demonstrated that all four enzymes exhibit PNPOx activity using both PNP and PMP substrates to produce PLP, with no unusual substrate specificity. All seven members of the annotated F420-dependent PNPOx family from Mtb were expressed in E. coli host cells, resulting in soluble expression of five proteins. In vitro binding assays revealed that these proteins bind specifically to coenzyme F420, with no apparent binding to either FMN or FAD. Activity assays clearly showed that these enzymes do not exhibit the annotated PNPOx activity. A previous research conducted in our laboratory has identified pteridine-like compounds (i.e. folate and its derivatives) as potential substrates for this family of enzymes. Based on this observation, a combination of thermal shift assays and enzymatic activity experiments were designed and performed. The results showed that two members of the annotated F420-dependent PNPOx family, Rv1875 and Rv3369, could catalyse the reduction of pteridine compounds in the presence of reduced F420. Although further investigation is required, these observations are the first reports of enzymatic activity for members of the mis-annotated F420-dependent PNPOx enzyme family. This research provides a comprehensive view of the classical FMN-dependent PNPOx enzymes from various mycobacterial species. It also sheds light on the cofactor specificity and potential physiological substrate(s) for members of the mis-annotated F420-dependent PNPOx enzymes. The results of this research could potentially pave the way for better understanding of the roles and importance of coenzyme F420 in the adaptive metabolic processes in Mtb.