Genetic, structural, and functional analysis of mutations causing methylmalonyl-CoA epimerase deficiency

Human methylmalonyl-CoA epimerase (MCEE) catalyzes the interconversion of D-methylmalonyl-CoA and L-methylmalonyl-CoA in propionate catabolism. Autosomal recessive mutations in MCEE reportedly cause methylmalonic aciduria (MMAuria) in eleven patients. We investigated a cohort of 150 individuals suffering from MMAuria of unknown origin, identifying ten new patients with mutations in MCEE. Nine patients were homozygous for the known nonsense mutation p.Arg47* (c.139C>T), and one for the novel missense mutation p.Ile53Arg (c.158T>G). To understand better the molecular basis of MCEE deficiency, we mapped p.Ile53Arg, and two previously described patient mutations p.Lys60Gln and p.Arg143Cys, onto our 1.8 Å structure of wild-type (wt) human MCEE. This revealed potential dimeric assembly disruption by p.Ile53Arg, but no clear defects from p.Lys60Gln or p.Arg143Cys. Functional analysis of MCEE-Ile53Arg expressed in a bacterial recombinant system as well as patient-derived fibroblasts revealed nearly undetectable soluble protein levels, defective globular protein behavior, and using a newly developed assay, lack of enzymatic activity - consistent with misfolded protein. By contrast, soluble protein levels, unfolding characteristics and activity of MCEE-Lys60Gln were comparable to wt, leaving unclear how this mutation may cause disease. MCEE-Arg143Cys was detectable at comparable levels to wt MCEE, but had slightly altered unfolding kinetics and greatly reduced activity. We solved the structure of MCEE-Arg143Cys to 1.9 Å and found significant disruption of two important loop structures, potentially impacting surface features as well as the active-site pocket. These studies reveal ten new patients with MCEE deficiency and rationalize misfolding and loss of activity as molecular defects in MCEE-type MMAuria.


Introduction
Propionyl-CoA is the common degradation product from branched-chain amino acids, oddchain fatty acids, and the side chain of cholesterol. The propionate catabolic pathway serves to funnel propionyl-CoA into the tricarboxylic acid (TCA) cycle for use as cellular energy sources through oxidative phosphorylation. Located at the centre of this pathway, methylmalonyl CoA epimerase (MCEE) catalyzes the epimerization of D-methylmalonyl-CoA, generated from propionyl-CoA by propionyl-CoA carboxylase (PCC), to form Lmethylmalonyl-CoA, subsequently converted into succinyl-CoA by methylmalonyl-CoA mutase (MUT) for entry into the TCA cycle.
Isolated methylmalonic aciduria (MMAuria), an inborn error of organic acid metabolism, is typically caused by deficiency of MUT or by a defect in the transport or processing of its organometallic cofactor, adenosylcobalamin. However, mutations in the human MCEE gene (OMIM #251120) have been identified in eleven cases of atypical MMAuria (1)(2)(3)(4)(5)(6). For two patients, coincidental mutations in the SPR gene causing sepiapterin reductase deficiency sufficiently explained their clinical symptoms (2,4), while two others have been described as asymptomatic (3), leaving the clinical importance of MCEE deficiency in doubt. The majority of patients with MCEE deficiency (seven including those with sepiapterin reductase deficiency) are homozygous for the stop-gain nonsense mutation c.139C>T (p.Arg47*)(1-6); the missense mutations p.Lys60Gln and p.Arg143Cys (3) and a splicing mutation (c.379-644A>G) (5) have also been identified, but the functional relevance of these missense mutations remains unclear.
In the human genome, MCEE is one of six proteins belonging to the vicinal oxygen chelate (VOC) superfamily, which include also glyoxalase I (GLO1 gene, GLOD1 protein), 4hydroxyphenylpyruvic acid dioxygenase (HPD, GLOD3), 4-hydroxyphenylpyruvic acid dioxygenase-like (HPDL, GLOXD1), and glyoxalase domain-containing 4 (GLOD4) and 5 (GLOD5). VOC members are metalloenzymes highly divergent in sequence and biological functions, but universally share the use of the β α β β β structural motif (also known as the glyoxylase fold) to build a divalent metal-containing active site (7,8). VOC enzymes catalyze a range of chemical reactions including isomerization, epimerization, oxidative C-C bond cleavage and nucleophilic substitution (9). The active-site divalent metal is used to bind the reaction substrate, intermediates or transition states in a bidentate fashion (8). To date, only structures from bacterial MCEE orthologues (Propionibacterium shermanii and Thermoanaerobacter tengcongensis) have been reported (10,11).
In this article, we conducted an in depth investigation of MCEE deficiency at the gene and protein levels. From a cohort of 150 patients with MMAuria of unknown etiology, we identified ten new patients with mutations on the MCEE gene including a novel missense mutation. We determined the crystal structure of human MCEE of the wild-type as well as p.Arg143Cys variant proteins. We further characterized protein expression and enzyme activity for the three known MCEE missense mutations associated with disease. Our study provides a molecular explanation for the biochemical defects associated with the missense mutations.

Identification of ten new MCEE patients with methylmalonic aciduria
Thus far, 11 cases of MMAuria have been identified due to mutations in the MCEE gene (1)(2)(3)(4)(5)(6). We screened fibroblast cell lines taken from 150 patients with mild but clear MMAuria who could not be assigned to a cobalamin class of defect. From this cohort, we identified ten patients from nine families with mutations in MCEE (Table 1). All patients are homozygous for their respective mutations, of which nine out of ten harbour the previously described c.139C>T (p.Arg47*) nonsense mutation. This remains by far the most common mutation identified in MCEE deficiency, with 16 out of 21 patients homozygous for this allele. One patient in our cohort harboured c.158T>G (p.Ile53Arg), a novel missense mutation that is not found in the ExAC database (>120,000 alleles) (12). We did not observe either c.178A<C (p.Lys60Gln), previously found in one patient in the homozygous state, or c.427C>T (p.Arg143Cys), previously found in two patients in the heterozygous state without an apparent second mutation (3).
In our cohort, disease onset ranged from 1 month old to 2.5 years of age, while from two patients we had no information. Clinical symptoms were variable but usually mild, and no patient was responsive to vitamin B 12 treatment. At least three patients presented following an intercurrent illness, while three presented with metabolic acidosis and/or hypoglycemia. In addition, elevated levels of other metabolites typical for a block in the propionate degradation pathway, such as 2-methylcitrate, propionylcarnitine and 3-hydroxypropionate, were documented in most patients. Investigation in patient fibroblasts revealed mildly decreased propionate incorporation (Table 1), which did not increase upon addition of hydroxocobalamin.

Structural features of human MCEE
We performed structural biology studies to establish the molecular basis of disease-causing mutations on the human MCEE protein (hMCEE). As a first step, we determined the crystal structure of wild-type (wt) protein (hMCEE WT ) to 1.8 Å resolution (Table 2), as part of a wider effort that also generated crystal structures of three other human VOCs, namely hHPD (PDB:  Fig. S1, the human VOCs display versatility in the way that four GLOD motifs are assembled, at the gene or protein level, to give rise to a minimal functional unit harbouring two metal-binding active sites. In the case of hMCEE, two β α β β β motifs pack side-by-side to form an 8-stranded sheet that completes the active site for one protomer (Fig. 1A). Crystallographic (supplemental Fig. S1) and solution data (supplemental Fig. S2) show that hMCEE is dimeric, in agreement with bacterial orthologs (10,11). The VOC members are so called because a divalent metal per active site can bind the substrate, intermediates or transition states in a bidentate fashion (8).

Structural mapping of MCEE mutations
Both the reported p.  Fig. S3). Loss of almost the entire protein is therefore the likely cause of enzymatic dysfunction due to this mutation, assuming there is residual mRNA following nonsense-mediated decay.
By contrast, the molecular dysfunction due to the missense mutations is less clear (Fig. 1C).
In our hMCEE structure, Ile53 is located at the dimeric interface, making hydrophobic contacts with Gly168 and Val169 from the loop region connecting strands ). An Ile-to-Arg at this position likely interferes with proper dimeric assembly, and is predicted by FoldX (13) to have severely reduced stability (ΔΔG 9.53 kcal/mol). By contrast, Lys60 and Arg143 occupy amino acid positions that are more variable. Position 60 is only occupied by lysine in 15% of MCEE homologs while position 143 is 60% occupied by arginine. Both residues are surface exposed and not directly involved in the dimeric interface and active-sites of both subunits ( Fig. 1D), consistent with a FoldX prediction of no effect for p.Lys60Gln (ΔΔG 0.3 kcal/mol), and moderately reduced stability for p.Arg143Cys (ΔΔG 2.26 kcal/mol).

Characterization of MCEE mutations in recombinant and patient cells
To validate our structural interpretation of mutations, we performed expression studies in E.
coli and human cells. When expressed in E. coli ( Fig revealed wt protein to be well expressed, while hMCEE containing p.Lys60Gln and p.Arg143Cys were detectable at only slightly lower levels (63 ± 17% and 74 ± 26% of wt, respectively) (Fig. 3B). However, hMCEE containing p.Ile53Arg had very low levels of detectable protein (6 ± 1% of wt), similar to empty vector (4 ± 2% of wt) (Fig. 3B). Thus, consistent with recombinant studies, it appears that p.Ile53Arg causes an inability to fold correctly.

Structure of human MCEE p.Arg143Cys variant
We determined the crystal structure of the p.Arg143Cys variant protein (hMCEE R143C ) to 1.9 Å resolution (Table 2), to directly inspect the atomic environment of the substitution (Fig. 4).
While hMCEE R143C superimposes well overall with hMCEE WT (Cα-RMSD 0.278 Å), significant main-chain displacement was clearly observed in the loop region connecting helix  In the hMCEE R143C structure, the substituted Cys143 residue generated more mobility and disorder within the loop

Biochemical characterization of MCEE mutations
We developed a radioactive HPLC-based assay to assess MCEE activity of wt and variants.
This assay follows the production and separation of [ 14 C]-methylmalonic acid and [ 14 C]succinic acid from [ 14 C]-propionic acid following addition of [ 14 C]-propionyl-CoA to fibroblast cell lysates, as depicted in Fig. 5A. Using UV detection, we were able to detect separated propionic acid, methylmalonic acid and succinic acid following HPLC analysis (supplemental

Conclusions
The identification of an additional ten patients with MCEE deficiency adds more information toward the debate of whether deficiency of this protein does indeed cause disease and not just elevated methylmalonic acid levels, and confirms that complete MCEE deficiency, despite low penetrance, may lead to an acute clinical phenotype with metabolic crisis resembling classical organic acidurias, similar to e.g. 3-methylcrotonyl-CoA carboxylase deficiency (14). Our combined structural, biophysical and enzymatic assessment of MCEE defects confirm the importance of this protein within the propionate degradation pathway. 8 While protein-destabilizing mutations (e.g. p.Ile53Arg) explains enzymatic defects more readily, mutations away from the active-site (e.g. p.Arg143Cys) could still cause a loss of activity, although the underlying mechanism needs further clarification. In regards to p.Lys60Gln, however, we could identify no defect conferred to the protein, leaving the molecular mechanism and its potential to cause disease in doubt.

Reagents
Unless otherwise noted, all compounds were obtained from Sigma-Aldrich (Buchs SG, Switzerland) and were reagent grade or better.

Size Exclusion Chromatography
Size exclusion chromatography was performed as described in (21).

Nano-Differential Scanning Fluorimetry
Melting curves for the wt protein and mutants were obtained via detection of changes in light scattering using the Prometheus NT.48. Protein concentration was kept at 100 µM in 50 mM HEPES pH 7.5, 500 mM NaCl, 0.5 mM TCEP, 5% glycerol and a melt gradient of 1 degree per minute 20°C to 95°C was used.

Patient Characterization (genotyping, propionate incorporation)
Skin fibroblasts were taken from patients with biochemical and clinical evidence of methylmalonic aciduria, referred to our institution for diagnostic purposes. The study has been approved by the ethics commission of the Canton of Zurich, Switzerland (application no. 2014-0211). Genomic DNA and RNA extraction, as well as sequencing and propionate incorporation were performed as previously described (21). The nomenclature of the mutations is based on the cDNA sequence NM_032601.3. Nucleotide numbering uses +1 as the A of the ATG translation initiation codon in the reference sequence, with the initiation codon as codon 1.

Transfection, Immunoblotting and Enzymatic Assay
A DNA fragment encompassing the entire coding sequence of wild-type MCEE was cloned into pcDNA3-CT10HF-LIC using LIC cloning. The sequence was as given by NM_032601.3, except for c.311T>G (p.Lue104Arg), whereby c.311G is the more common allele, (12). Sitedirected mutagenesis was carried out on this construct using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) as described in the manufacturer's instructions, using forward and reverse primers (Microsynth, Balgach, Switzerland) and confirmed by Sanger sequencing. Control (BJ, CRL-2522, ATCC) or patient (carrying homozygous MCEE mutation c.139C>T, p.Arg47*) fibroblasts were transiently transfected with 10 µg wild-type or mutant MCEE constructs, with or without 10 µg MUT in pTracer (22) for the enzymatic assay, using electroporation (23). Cells were grown in Dulbecco's Modified Eagle Medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and antibiotics (GE Healthcare), as previously described (24) and harvested by trypsinization 48 hr after electroporation, washed twice with HBSS (Gibco) and either frozen at −20°C or processed directly.
For the enzymatic assay, fresh or frozen cell pellet was lysed in buffer including 12 mM Tris-HCl, pH 8.0 and 1mM DTT with sonication twice at amplitude 1.5 for 15 seconds using the microprobe of an XL-2000 sonicator (Microson, Qsonica Newtown, CT). Following lysis, protein concentration was determined by the Lowry method. The incubation mixture contained 300-500 µg cell protein, reaction buffer (100 mM Tris-HCl, pH 8.0; 6 mM MgCl 2 ; 3.15 mM ATP; 100 mM KCl; 3 mM DTT), 1 mM propionyl-CoA mix (1 mM propionyl-CoA and 7 µM [ 14 C]-propionyl-CoA at 55 mCi/mmol, Anawa, Switzerland) and 50 uM adenosylcobalamin and reaction time was 60 minutes at 37°C. The reaction was stopped by adding 0.5M KOH, samples were then re-incubated for 15 minutes at 37°C to hydrolyze CoA derivatives, neutralized by adding 0.5 N perchloric acid (Merck), and spiked with 0.05% succinic acid, 0.018% methylmalonic acid and 0.018% propionic acid in order to visualize peaks during HPLC separation. Samples were centrifuged to remove precipitate, and supernatant was injected into an Aminex HPX-87H Ion Exclusion column (300 × 7.8 mm2; Hform, 9 μ m, Bio-Rad), and organic acids separated by elution with 0.5 mM H 2 SO 4 at 30°C using a flow rate of 0.4 ml/min. Retention times were 13-15 minutes for methylmalonic acid, 17-19 minutes for succinic acid, and 26-28 minutes for propionic acid (supplemental Fig. S5  and 6), visualized at 210 nm using a UV detector. Fractions covering the methylmalonic acid and succinic acid peaks were collected and [ 14 C]-methylmalonic acid and [ 14 C]-succinic acid quantified in a Tri-Carb C1 900TR scintillation spectrometer (PerkinElmer,Waltham, MA, USA) with OptiphaseHiSafe 2 counting cocktail (PerkinElmer).    α -helices that are part of the GLOD motif are red, and regions that connect GLOD motifs are yellow. B. Active-site architecture showing the divalent metal and metal coordinating residues. Pink sphere: cobalt. C. On the hMCEE monomer, residues that are mutated in disease are labeled in orange, active-site residues are labeled in black. D. hMCEE physiological dimer with the second subunit represented as grey space fill. Mutations are labeled in orange and designated as belonging to chain (monomer) A or B.