Methylmalonic aciduria and homocystinuria, cblC type, is the most common inborn error of cellular vitamin B12 metabolism. We previously showed that the protein carrying the mutation responsible for late-onset cblC (MMACHC-R161Q), treatable with high dose OHCbl, is able to bind OHCbl with wild-type affinity, leaving undetermined the disease mechanism involved [Froese et al., Mechanism of responsiveness, Mol. Genet. Metab. (2009).]. To assess whether the mutation renders the protein unstable, we investigated the thermostability of the wild-type and mutant MMACHC proteins, either unbound or bound to different cobalamins (Cbl), using differential scanning fluorimetry. We found that MMACHC-wt and MMACHC-R161Q are both very thermolabile proteins in their apo forms, with melting temperatures (Tm) of 39.3 ± 1.0 and 37.1 ± 0.7 °C, respectively; a difference confirmed by unfolding of MMACHC-R161Q but not MMACHC-wt by isothermal denaturation at 35 °C over 120 min. However, with the addition of OHCbl, MMACHC-wt becomes significantly stabilized (ΔTm max = 8 °C, half-maximal effective ligand concentration, AC50 = 3 μM). We surveyed the effect of different cobalamins on the stabilization of the wild-type protein and found that AdoCbl was the most stabilizing, exerting a maximum increase in Tm of ∼16 °C, followed by MeCbl at ∼13 °C, each evaluated at 50 μM cofactor. The other cobalamins stabilized in the order (CN)2Cbi > OHCbl > CNCbl. Interestingly, the AC50’s for AdoCbl, MeCbl, (CN)2Cbi and OHCbl were similar and ranged from 1–3 μM, which compares well with the Kd of 6 μM for OHCbl [Froese et al., Mechanism of responsiveness, Mol. Genet. Metab. (2009).]. Unlike MMACHC-wt, the mutant protein MMACHC-R161Q is only moderately stabilized by OHCbl (ΔTm max = 4 °C). The dose–response curve also shows a lower effectivity of OHCbl with respect to stabilization, with an AC50 of 7 μM. MMACHC-R161Q showed the same order of stabilization as MMACHC-wt, but each cobalamin stabilized this mutant protein less than its wild-type counterpart. Additionally, MMACHC-R161Q had a higher AC50 for each cobalamin form compared to MMACHC-wt. Finally, we show that MMACHC-R161Q is able to support the base-off transition for AdoCbl and CNCbl, indicating this mutant is not blocked in that respect. Taken together, our results suggest that protein stability, as well as propensity for ligand-induced stabilization, contributes to the disease mechanism in late-onset cblC disorder. Our results underscore the importance of cofactor stabilization of MMACHC and suggest that even small increases in the concentration of cobalamin complexed with MMACHC may have therapeutic benefit in children with the late-onset, vitamin responsive cblC disease.

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The valAB locus of Francisella novicida has previously been found to be highly similar at the deduced amino acid level to msbA lpxK of Escherichia coli. Both ValA and MsbA are members of the superfamily of ABC transporters, and they appear to have similar functions. In this study we describe the isolation of a temperature-sensitive valAB locus. DNA sequence analysis indicates that the only changes to the ValAB deduced amino acid sequence are changes of S453 to an F and T458 to an I in ValA. E. coli strains defective in msbA and expressing temperature-sensitive ValA rapidly ceased growth when shifted from a permissive temperature to a restrictive temperature. After 1 h at the restrictive temperature, cells were much more sensitive to deoxycholate treatment. To test the hypothesis that ValA is responsible for the transport or assembly of lipopolysaccharide, we introduced gseA, a Kdo (3-deoxy-D-manno-octulosonic acid) transferase from Chlamydia trachomatis, into a strain with a temperature-sensitive valA allele and a nonfunctional msbA locus. These recombinants were defective in cell surface expression of the chlamydial genus-specific epitope within 15 min of a shift to the nonpermissive temperature. Also, there was enhanced association of the epitope with the inner membrane after a shift to the nonpermissive temperature. Thus, we propose that ValA is involved in the transport of lipopolysaccharide to the outer membrane.We have recently discovered a genetic locus in Francisella novicida that contains an apparent operon consisting of two open reading frames, valAB (13). F. novicida strains thought to harbor mutations in valAB are defective in growth in macrophages and exhibit increased sensitivity to serum and deoxycholate, thus suggesting compromised outer membrane integrity. These two genes show high identity at the deduced amino acid level to the Escherichia coli genes msbA and lpxK, respectively.Interestingly, the msbA gene was originally identified as a multicopy suppressor of the E. coli htrB gene (8). Mutants having lesions in htrB exhibit an unusual phenotype whereby growth and viability of mutants at temperatures above 32.5°C in rich media are impaired. The gene product of htrB has been shown to be a Kdo (3-deoxy-D-manno-octulosonic acid)-dependent acyl transferase involved in lipopolysaccharide (LPS) biosynthesis (3). We have previously shown that, similar to msbA, valAB can suppress mutations in htrB, which suggests that both gene products may have similar functions related to LPS expression. Recent work has demonstrated that lpxK (previously called orfE) encodes a lipid A 4Ј-kinase (6).The presumptive gene products ValA and MsbA are members of the superfamily of ATP binding cassette (ABC) transporters. ABC transporters are a large family of integral membrane proteins that are responsible for the uptake or efflux of a wide variety of both proteinaceous and nonproteinaceous substrates (4). In several bacterial systems, the involvement of ABC transporters in the export of capsular polysaccharide and the tra...