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Fungal infections pose an urgent threat to human health and are responsible for approximately 1.6 million deaths annually. Current therapeutic options for life-threatening mycoses, such as those caused by Aspergillus fumigatus, Candida albicans or Cryptococcus neoformans, are severely limited and even under antifungal treatment these infections have mortality rates of ~50%. To improve patient outcomes there is a critical need for development of novel antimycotic drugs. This urgency is further exacerbated by the pharmacological weaknesses of current chemotherapeutics, surges in susceptible patient groups and rising antifungal resistance. Fungal pathogenic potential is strongly dependent on metabolic versatility. Pathways required for cellular metabolism and nutrient homeostasis in host tissues are fundamental for infection and represent exciting targets for drug discovery. The fungal cobalamin-independent methionine synthase (metH), which belongs to a different enzyme class to the cobalamin-dependent version found in humans, forms a junction between two critical primary metabolic pathways: the transsulfuration and one-carbon cycles. The enzyme has been shown to be essential for the viability and/ or virulence of A. fumigatus and other clinically relevant fungal species indicating it has potential as a fungal specific target with broad applications. However, the reason why MetH is indispensable for fungi beyond methionine auxotrophy, has so far not been identified. To improve the likelihood of success in the drug discovery process and fully exploit the enzyme's potential as an antifungal target this thesis investigated the mechanistic basis of MetH's essentiality in A. fumigatus. Phenotypic analyses using point-mutated versions of MetH revealed that enzymatic activity converting homocysteine to methionine is indispensable for A. fumigatus viability. Overexpression of genes that prevent accumulation of the potentially toxic substrate homocysteine did not rescue growth, implying that its accumulation is not responsible for the enzyme's essentiality. Supplementation of the growth media with several metabolites which are potentially depleted in the absence of MetH's enzymatic activity restored fungal viability with very limited growth. This suggested that methionine synthases' essentiality is explained by the depletion of critical molecules. Metabolomics analyses indicated that suppressing metH reduces cell energetics and phenotypic experiments confirmed that this was the additional cause of MetH essentiality in A. fumigatus, as supplementation with compounds selected to enhance energetics was able to fully rescue growth of the fungus in the absence of methionine synthase enzymatic activity. Taken together, our results suggest that cellular energetics is deleteriously disrupted in MetH's absence, supporting methionine synthase's value as an antifungal drug target. As conditions encountered, and consequently fungal metabolic requirements, may vary significantly throughout infection it is crucial to validate targets in established infections. To ascertain the relevance of MetH for fungal growth in various developmental stages we took advantage of the metH_tetOFF mutant, which expresses metH under the control of the regulatable tetOFF system, whereby the addition of doxycycline (Dox) shuts down expression of the gene. Modulation of metH expression in vitro revealed that it is required for growth of conidia, germlings and hyphae in a fungistatic manner. The genetic model was optimised for use in murine and Galleria mellonella models of established invasive aspergillosis. These models showed that downregulation of metH reduces A. fumigatus virulence in established infections at a level comparable to downregulating expression of the target of the azoles, the lanosterol 14-a-demethylase, making methionine synthase an attractive antifungal drug target. Following validation of MetH as a promising antifungal target this work investigated whether the enzyme was druggable and assayable. In this study we aimed to use complimentary approaches to initiate the early stages of drug development: a structure-based virtual screening for the directed design of binding fragments and optimisation of a simple, cost-effective enzymatic assay suitable for high-throughput screening. To facilitate computational analyses the structure A. fumigatus MetH was predicted using the previously crystallised, and highly similar, C. albicans version of the enzyme. In silico comparisons determined structural differences between the substrate binding sites of human and fungal methionine synthases, which could guide the production of novel fungal specific inhibitors with reduced off target binding. Molecular docking of ligands from the Maybridge Ro3 fragment library identified fragments with a high probability to specifically bind regions crucial for the fungal MetH's enzymatic activity. Finally, the expression and purification of soluble MetH in Escherichia coli was achieved, which allowed optimisation of an economic method of measuring appearance of one of the products of MetH's enzymatic reaction. This enzymatic assay provides a promising tool by which to measure the ability of potential drugs to inhibit methionine synthase activity during the early stages of drug development. Overall, our results support MetH as an attractive druggable, specific and broad-spectrum antifungal drug target that is relevant to the establishment and progression of invasive disease. The insights gained in this work provides the opportunity to harness the full potential of methionine synthase as an antimycotic target in A. fumigatus and provides tools for the interrogation and validation of other targets in in vivo models of established invasive pulmonary aspergillosis.