Abstract
Human ATP:cob(I)alamin adenosyltransferase (ATR) is a mitochondrial enzyme that catalyzes an adenosyl transfer to cob(I)alamin, synthesizing 5′-deoxyadenosylcobalamin (AdoCbl) or coenzyme B12. ATR is also a chaperone that escorts AdoCbl, transferring it to methylmalonyl-CoA mutase, which is important in propionate metabolism. Mutations in ATR lead to methylmalonic aciduria type B, an inborn error of B12 metabolism. Our previous studies have furnished insights into how ATR protein dynamics influence redox-linked cobalt coordination chemistry, controlling its catalytic versus chaperone functions. In this study, we have characterized three patient mutations at two conserved active site residues in human ATR, R190C/H, and E193K and obtained crystal structures of R190C and E193K variants, which display only subtle structural changes. All three mutations were found to weaken affinities for the cob(II)alamin substrate and the AdoCbl product and increase KM(ATP). 31P NMR studies show that binding of the triphosphate product, formed during the adenosylation reaction, is also weakened. However, although the kcat of this reaction is significantly diminished for the R190C/H mutants, it is comparable with the WT enzyme for the E193K variant, revealing the catalytic importance of Arg-190. Furthermore, although the E193K mutation selectively impairs the chaperone function by promoting product release into solution, its catalytic function might be unaffected at physiological ATP concentrations. In contrast, the R190C/H mutations affect both the catalytic and chaperoning activities of ATR. Because the E193K mutation spares the catalytic activity of ATR, our data suggest that the patients carrying this mutation are more likely to be responsive to cobalamin therapy.
Highlights
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Since the E193K mutation spares the catalytic activity of ATR, our data suggest that patients carrying this mutation are more likely to be responsive to cobalamin therapy
ATR (Fig. 2D), indicating either greatly weakened affinity or the presence of bound 5coordinate cob(II)alamin. To distinguish between these possibilities, cob(II)alamin (100 μM) was mixed with E193K ATR (500 μM trimer) and ATP (2 mM) and the unbound ligands were separated by centrifugation using a 10 kDa cut-off filter
Summary
The ATR mutants were purified in comparable yield to wild-type protein and in >95% purity (Fig. 2A). The crystal structure of ATP bound to R190C ATR (Table 2) was solved by molecular replacement at 1.5 Å resolution, using the structure of wild-type human ATR with ATP (PDB code: 2IDX) [24] An alignment of these two structures showed that the R190C mutation turn, the higher KD values for cob(II)alamin (in the presence of ATP) and AdoCbl (± PPPi) (Table 1). These data are consistent with a role for ATP in facilitating cobalamin binding by stabilizing the active site architecture. Despite the 52-fold increase in the KM(ATP) value to 235 ± 16 μM, the reaction rate of E193K
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