Functional and Structural Characterisation of Bacterial ATP-grasp Ligases

Abstract

Enzymes are extremely important in biology, acting as protein catalysts to accelerate otherwise kinetically unfavourable biochemical reactions. One enzyme group of interest that is present throughout all domains of life is the ATP-grasp superfamily, consisting of enzymes which each possess a structurally conserved motif known as the “ATP-grasp” fold. The ATP-grasp fold accommodates the cofactor ATP which is used by these enzymes to catalyse a specific biochemical reaction, most commonly involving the ligation of two molecular substrates. Despite sharing this feature, each ATP-grasp ligase is highly selective for a particular substrate pair. Due to the diverse nature of ATP-grasp ligases they have garnered interest for several reasons, primarily being i) to characterise new mechanistic features of ATP-grasp ligases ii) to identify ATP-grasp ligases with new catalytic activities and biological functions, and iii) to apply this knowledge to exploit ATP-grasp ligases for useful applications – for example as drug targets. These avenues of investigation form the basis of this thesis, which involves the structural and functional characterisation of four bacterial ATP-grasp ligases to understand their catalytic activity, mechanism, and biological functions/applications. To this end, a combination of X-ray crystallography and a newly developed enzyme activity assay were employed as core techniques. First, the essential bacterial enzyme and antibiotic target ᴅ-alanine–ᴅ-alanine ligase (Ddl) was investigated to understand how it is activated by monovalent cations (MVCs), a 60 year old question. This work provided new mechanistic insight, determining that Ddl is selectively activated by potassium to improve catalytic efficiency, and revealed this property may be shared by other members of the ATP-grasp superfamily. Additionally, to develop more effective inhibitors of Ddl, a series of N-acyl-substituted sulfamide compounds based on the ATPscaffold were designed to span both the ATP and ᴅ-alanine binding pockets of Staphylococcus aureus Ddl (SaDdl). These compounds were ranked by in silico docking to SaDdl, synthesised, and assayed for inhibitory activity against SaDdl. While no potent inhibitors of SaDdl were identified by this approach, it provides useful insight into the development of future bisubstrate inhibitors targeting Ddl. The second enzyme investigated was a previously uncharacterised ATP-grasp ligase of the human pathogen Staphylococcus aureus. The genetic localisation of the corresponding gene indicated a possible role in methionine metabolism, a key metabolic pathway of Staphylococci of interest for antibiotic development. By screening the activity of this enzyme, it was identified as ʟ-aspartate–ʟ-methionine ligase (LdmS), preferentially catalysing the formation of the dipeptide product ʟ-aspartyl–ʟ-methionine. A combination of X-ray crystallography, in silico docking and site-directed mutagenesis revealed the determinants of this unique substrate specificity and provided understanding of the LdmS catalytic mechanism. The application of LdmS for enzymatic dipeptide synthesis was also investigated. Finally, two homologous enzymes of Escherichia coli, YgiC and YjfC, were characterised to gain insight into their catalytic function. These enzymes share similarity to an ATP-grasp ligase that produces a unique conjugate of the antioxidant tripeptide glutathione with spermidine, however YgiC and YjfC are unable to catalyse this reaction. By solving the crystal structure for each enzyme, the structural basis for this discrepancy was revealed, with differences in amino acid residues forming the substrate binding pocket the likely cause. Probing activity with model substrates demonstrated YgiC and YjfC catalyse the formation of peptide-spermidine conjugates, suggesting that both enzymes are involved in the synthesis of a novel Escherichia coli metabolite.