Investigating the structure and function of CobH and CobB, two consecutive enzymes in the biosynthesis of cobalamin

Cobalamin (vitamin B12) is the anti-pernicious anaemia factor that is made exclusively by certain prokaryotes. It is constructed along one of the most complex biosynthetic pathways found in nature, involving around 30 enzymes-mediated steps. In the aerobic cobalamin biosynthesis pathway, the central cobalt ion of the corrin component of cobalamin is inserted into a comparatively late pathway intermediate called hydrogenobyrinic acid-a,c-diamide (HBAD) to give cobyric acid. This section of the pathway appears to be very sensitive to feedback inhibition and hence there is a lot of interest in how HBAD is made. Surprisingly, there is no structural information about the enzyme that makes HBAD, CobB. This enzyme takes hydrogenobyrinic acid and amidates the a and c side chains to give HBAD. It is thought that CobB interacts closely with the preceding enzyme in the pathway, CobH, which is known to bind its product, hydrogenobyrinic acid (HBA), very tightly. In this project, attempts have been made to understand the structure and function of CobH and CobB through the application of protein crystallisation and X-ray crystallography. It is thought that CobB may interact with CobH in such way as to release the product and allow its amidation. Herein, the Allochromatium vinosum CobH and CobB have been purified and entered into a broad range of different crystal screens. The interaction between the two proteins has also been investigated using standard biochemical techniques. Although the CobB enzyme was observed to be more stable than CobB from other organisms, the CobH was found to be prone to proteolysis. The work suggests that the structure determination of CobB should be possible