Structural and Biophysical Analysis of Enzymes involved in the Amidation Process in the Cobalamin (Vitamin B12) Pathway

Cobalamin, more commonly known as vitamin B12, is a highly complex molecule whose biosynthesis in bacteria and archaea involves around 30 enzyme-mediated steps. The biosynthesis of vitamin B12 occurs by one of two distinct pathways, which are referred to as the aerobic and the anaerobic routes. The work described in this thesis relates to the aerobic pathway, and is mainly focussed on two enzymes responsible for the amidation of the corrin ring component of cobalamin, CobB which is the enzyme responsible for the amidation of HBA to its product HBAD and CobQ which amidates cobyrinic acid a, c-diamide to cobyrinic acid. These enzymes amidate the peripheral carboxylic acid sidechains of the corrin moiety, and produce hydrogenobyrinic acid (HBA) and adenosylcobyrinic acid a, c-diamide, respectively. CobB, amidates the a and c side chains attached to C2 and C7 of the corrin ring, of HBA. The product of this multi-functional enzyme is initially hydrogenobyrinic acid c- monoamide (HBAM) followed by hydrogenobyrinic acid a, c-diamide (HBAD).

The gene encoding CobB was cloned from Brucella melitensis, overexpressed and its encoded protein purified. After crystallisation of the B. melitensis CobB, the first X- ray crystal structure of this enzyme was solved using selenomethionine labelled protein to 1.6 Å resolution. Further high resolution structures were obtained containing substrate and co-factors.

To complement the structural information, kinetic data was acquired for the binding of CobB with its substrates, using stopped flow analysis. This kinetic approach helped identify a two-phase binding process, indicating that the initial HBA molecule binds quickly to the dimeric enzyme, whilst the second HBA molecule binds at a much slower rate. Collectively, the binding data suggest that CobB undergoes a conformational change upon binding the first HBA substrate that alters the binding affinity of the second HBA-binding site.

3

CobH, the enzyme prior to CobB in the cobalamin pathway, is known to bind its product (HBA) very tightly, which poses the question as to how CobB interacts with CobH in order to access its substrate. To investigate if CobB and CobH interact a model was constructed to determine if the two enzymes could form a complex that facilitates direct metabolite channelling. Evidence for direct metabolite transfer was obtained from fluorescence spectroscopy, where fluorescence emission was used to monitor changes associated with substrate transfer of HBA from CobH to CobB Co- crystallisation trials to obtain a complexed crystal structure of CobH/HBA/CobB was attempted, but this approach resulted only in the generation of CobH crystals.

CobQ, the second enzyme in the amidation process, was also produced recombinantly and purified. This enzyme amidates positions b, d, e and f on the corrin ring. Crystallisation trials were attempted, and a low resolution data set was collected (4.5 Å). However, the structure was not determined due to the poor quality of the data.

CobB and CobQ are key enzymes involved in the amidation process in the vitamin B12 pathway. To date there is no structural information in the literature as to how these enzymes function. Obtaining a crystal structure of either CobB or CobQ would enhance our knowledge on these multi-functional enzymes, enabling a clearer understanding on how they bind their substrates, and how these enzymes pass their product to the next enzyme in the vitamin B12 pathway.