Investigation of a novel intein-based Escherichia coli expression system for human methylmalonyl CoA mutase : a thesis presented to Massey University in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry
Human methylmalonyl CoA mutase (hMCM) is a 78 kDa homodimeric mitochondrial matrix enzyme. hMCM catalyses the conversion of 2R-methylmalonyl CoA to succinyl CoA in the metabolism of propionyl groups, and requires the vitamin B12
-derived cofactor adenosylcobalamin (AdoCbl). The mechanism of catalysis involves homolytic cleavage of AdoCbl's unusual C-Co bond, to generate radicals. Dysfunctional hMCM results in the rare, potentially fatal metabolic disorder methylmalonic acidemia. An experimentally determined structure of hMCM would add to the understanding of both the mechanism of catalysis and the molecular basis of some of the mutations underlying methylmalonic acidemia. The structure of the bacterial orthologue from Propionibacterium shermanii has been solved by x-ray crystallography, enabling the development of structural models of hMCM. Critical differences, however, between these two enzymes, mean that some regions of the models could be inaccurate. There is no x-ray crystal structure of hMCM. Purification of native hMCM for crystallization trials is complicated by ethical problems, low yields, and heterogeneity generated by the cofactor. To provide a more convenient source of pure, active human methylmalonyl CoA mutase for x-ray crystallography, an expression system for recombinant hMCM is required. Other researchers have expressed hMCM in Escherichia coli as (i) insoluble inclusion bodies, (ii) soluble fusion protein that cannot be separated efficiently from the fusion tag, or (iii) in low quantities. This research aimed to develop an E. coli expression system for the production of active human methylmalonyl CoA mutase, to enable x-ray crystallography structural studies. Based on the results of previous expression systems, four novel expression vectors were developed utilising the maltose binding protein and thioredoxin as solubility tags. It was hoped that conventional protease cleavage, to remove these solubility tags, could be circumvented by the use of intein-mediated cleavage. Intein-mediated cleavage was successful, and soluble active hMCM was recovered in low yields from a C-terminal thioredoxin solubility tag construct. hMCM was insoluble when expressed with MBP at the C-terminus.