Mechanism of vitamin B12-responsiveness in cblC methylmalonic aciduria with homocystinuria.

Patients with the cblC vitamin B(12) (cobalamin, cbl) disorder are defective in the intracellular synthesis of adenosylcobalamin and methylcobalamin and have combined homocystinuria and methylmalonic aciduria. While other vitamin B(12) disorders are treatable with high dose cyanocobalamin (CNCbl) or hydroxocobalamin (OHCbl), cblC patients respond well to OHCbl but not to CNCbl. Patient mutations were introduced into recombinant MMACHC (cblC) protein and the binding of CNCbl and OHCbl was examined. Three mutations were analyzed: G147D, associated with early onset, vitamin B(12) unresponsive disease; R161Q, associated with late onset disease that is highly responsive to OHCbl; and H122A, selected to test the hypothesis that H122 is central to a proposed vitamin B(12) binding motif on MMACHC. We report here that wild-type MMACHC binds both OHCbl and CNCbl with similar, tight affinity (K(d)=5.7 microM). We also report that MMACHC binds CNCbl in the base-off form, with the dimethylbenzimidazole (DMB) base of cobalamin displaced from coordination with the cobalt. In this form, wild-type MMACHC is able to reductively decyanate CNCbl to cob(II)alamin requiring only the presence of NADPH and FAD. We demonstrate that MMACHC with the G147D mutation is unable to bind either CNCbl or OHCbl, providing a straight forward explanation for the absence of response to either vitamin form. However, we show that MMACHC containing the R161Q mutation binds OHCbl with wild-type affinity, but is disturbed in binding CNCbl and has impaired decyanation. Finally, we show that H122A has reduced binding, but like R161Q, it binds OHCbl more tightly than CNCbl, suggesting that this histidine is not absolutely required for binding. These studies suggest that the ability of mutant MMACHC to respond to vitamin therapy depends on its ability to bind the vitamin with significant affinity, and for CNCbl, also on its ability to bind in the base-off form to facilitate reductive decyanation. These studies emphasize the continued use of OHCbl with cblC patients for maximum therapeutic effect.