| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 1999322 | Molecular Genetics and Metabolism | 2006 | 8 Pages |
ATP:cob(I)alamin adenosyltransferase (MMAB protein; methylmalonic aciduria type B) is an enzyme of vitamin B12 metabolism that converts reduced cob(I)alamin to the adenosylcobalamin co-factor required for the functional activity of methylmalonyl-CoA mutase. Mutations in the human MMAB gene result in a block in adenosylcobalamin synthesis and are responsible for the cblB complementation group of inherited vitamin B12 disorders. In this study, we examined the impact of several mutations, previously identified in cblB patients and clustered within a small, highly conserved region in MMAB. We confirmed mitochondrial expression of MMAB in human cells and showed that two mutations, R186W and E193K, were associated with absent protein by Western blot, while one, R191W, coupled with another point mutation, produced a protein in patient fibroblasts. Wild type MMAB and all four mutant proteins were stably expressed at high level as GST-fusion proteins, but only the R191W protein was enzymatically active. It showed an elevated Km of 320 μM (vs 6.8 μM for wild type enzyme) for ATP and 60 μM (vs 3.7 μM) for cob(I)alamin, with a reduction in kcat for both substrates. Circular dichroism spectroscopy revealed that three mutant proteins examined retained a α-helical structure as for the wild type protein. Characterization of MMAB will contribute to our understanding of cobalamin processing in mammalian cells and of disease mechanisms in the genetic disorders.
