Energetics of Interaction between the G-protein Chaperone, MeaB, and B12-dependent Methylmalonyl-CoA Mutase

Abstract

MeaB is an auxiliary protein that supports the function of the radical B 12 -dependent enzyme, methylmalonyl-CoA mutase, although its precise role is not understood. Mutations in the human homolog of MeaB, MMAA, lead to methylmalonic aciduria, an inborn error of metabolism that can be fatal. To obtain insights into the function of this recently discovered protein, we have characterized the entropic and enthalpic contributions to ⌬G assoc°f or complexation of MeaB (in the presence and absence of nucleotides) with methylmalonyl-CoA mutase (in the presence and absence of cofactor). The dissociation constant for binding of methylmalonyl-CoA mutase and MeaB ranges from 34 ؎ 4 to 524 ؎ 66 nM, depending on the combination of nucleotide and mutase form. Holomutase binds MeaB 15-fold more tightly when the nonhydrolyzable GTP analog, GMPPNP, is bound versus GDP. In contrast, the apomutase binds MeaB with similar affinity in the presence of either nucleotide. Our studies reveal that a large structural rearrangement accompanies interaction between these proteins and buries between ϳ4000 and 8600 Å 2 of surface area, depending on the combination of ligands in the active sites of the two proteins. Furthermore, we demonstrate that MeaB binds GTP and GDP with similar affinity (K d of 7.3 ؎ 1.9 and 6.2 ؎ 0.7 M, respectively at 20°C) and has low intrinsic GTPase activity (ϳ0.04 min ؊1 at 37°C), which is stimulated ϳ100-fold by methylmalonyl-CoA mutase. These studies provide insights into the energetics of interaction between the radical enzyme methylmalonyl-CoA mutase and MeaB, which are discussed.In recent years a number of auxiliary P-loop GTPases that function as chaperones in the assembly of metal cofactors in target enzymes have been described (1). Their GTPase activity was initially inferred from sequence analysis that revealed the presence of signature motifs associated with this superfamily (2) and include a Walker A or P-loop and Walker B motifs, an aspartate residue involved in Mg 2ϩ binding, and a GTP-binding (N/T)KXD sequence. Members of this family include UreG, which is involved in the assembly of the nickel-based metallocenter in urease (3, 4); HypB, which is required for nickel hydrogenases (5); CooC for CO dehydrogenase (6); and MeaB for B 12 -dependent methylmalonyl-CoA mutase (7). Although many of these proteins have since been demonstrated to possess low GTPase activity, their precise function in the assembly of their respective metalloprotein targets remains to be resolved.The most recently described member of this subfamily of GTPases is MeaB, which is strongly associated in bacterial operons with methylmalonyl-CoA mutase (8), a B 12 -dependent isomerase that catalyzes the rearrangement of methylmalonyl-CoA to succinyl-CoA (9, 10). An ortholog of MeaB, MMAA, is found in humans and is the locus of mutations associated with the cblA class of inborn errors of cobalamin disorders that leads to methylmalonic aciduria (11,12). In humans, the cblA gene product and methylmalonyl-CoA mutase are localized in the mitoc...

Visualization of a radical B 12 enzyme with its G-protein chaperone

“…This location provides a molecular explanation for the observation that chaperone GTPase activity is affected by binding to a target protein, as observed with MCM: MMAA/MeaB (33,39). Sizable regions of the GTPase active site are disordered in structures of MeaB and MMAA alone, and likely become ordered upon complex formation to assume the positions observed in IcmF.…”

IcmF Is a Fusion between the Radical B12 Enzyme Isobutyryl-CoA Mutase and Its G-protein Chaperone

“…8D). In comparison, MeaB alone exhibits a lower intrinsic GTPase activity (k cat ϭ 0.039 Ϯ 0.003 min Ϫ1 ), which is increased ϳ100-fold in the presence of MCM (15).…”

“…The isothermal titration calorimetric experiments were performed as described previously (14,15). Each experiment was performed in triplicate.…”

“…However, in IcmF, this motif is similar but not identical to that seen in ICM and MCM. First, a Gly 3 Ala/Ser change is found in IcmFs in the following Analysis of G-protein Domains-The P-loop GTPase domain has very high sequence similarity to MeaB from M. extorquens that was shown to be a chaperone for MCM (14,15). We designated it as MeaI to distinguish it from the MeaB-like chaperone.…”

“…MCM, in turn, influences the GTPase activity of MeaB increasing it by Ͼ100-fold. Hence, MCM exhibits GTPase-activating protein activity for MeaB (14,15). In addition, MCM modulates the affinity of MeaB for nucleotides.…”