Characterisation of the Enzymes Involved in the Anaerobic Biosynthesis of Benzimidazolylcobamides

Vitamin B12 is a complex metalloorganic cofactor involved in a variety of biochemical reactions that are essential for life. There are two routes by which its biosynthesis can be achieved: the oxygen independent (anaerobic) and oxygen dependent (aerobic), involving around 30 enzymatic steps. However, little biochemical characterisation has been conducted on the newly identified bza operon, which has been shown to encode enzymes capable of biosynthesising dimethylbenzamidizole (DMB), the α-ligand of vitamin B12. Furthermore, other bza-like operons have been linked to the production of cobamides with alternatively functionalised benzimidazole α-ligands (benzimidazolylcobamides). This thesis describes the biochemical investigation of the enzymes involved in these Bza and Bza-like pathways, aiming to provide insight into how they operate.

Three different BzaC enzymes identified in Bza and Bza-like pathways have been recombinantly produced, purified and biochemically characterised. This involved reconstitution of BzaCs activity with a benzimidazolylcobamide substrate, investigation of their reaction mechanisms, substrate specificity, inhibition and 3D structure. Furthermore, an interesting BzaE enzyme, which potentially belongs to the newly identified B12 dependent radical SAM family, identified in a Bza-like pathway has been investigated.

This work identified that BzaC can catalyse regioselective SAM dependent methyl transfer to 5(6)-hydroxybenzimidazolylcobamide with differences seen in percentage conversion of substrate to product across the BzaCs investigated. Assays containing 5(6)-hydroxybenzimidazole cobamides with differing upper ligands (cyano, hydroxo, adenosyl) identified that the enzymes prefer an adenosylated substrate. Furthermore, the highest performing BzaC, DtBzaC, was subject to kinetic analysis identifying a low micro molar affinity for its adenosylated substate (Km = 11 µM), together supporting BzaC's role in cobamide biosynthesis.

The 3D crystal structure of the DtBzaC dimer with SAH identified the SAM binding domain and in combination with substrate docking and structural comparison to similar enzymes allowed for a catalytic mechanism to be proposed, in which an active site histidine residue can deprotonate the substrate hydroxyl group, increasing its nucleophilicity, promoting SAM dependent methyltransfer.

In addition, biochemical analysis of DtBzaE identified that it can catalyse the radical rearrangement of adenosyl 5(6)-methoxybenzimidazolylcobamide likely forming a hydroxymethyl product (Ado[5(6)-CH2OH]Cba), deviating from the currently proposed reaction scheme for a BzaE enzyme.

Together the studies conducted in this thesis have contributed novel findings to the field, shifting the understanding of how these enzymes and pathway's function.