Ethylmalonyl-CoA Mutase from Rhodobacter sphaeroides Defines a New Subclade of Coenzyme B12-dependent Acyl-CoA Mutases*

Coenzyme B12-dependent mutases are radical enzymes that catalyze reversible carbon skeleton rearrangement reactions. Here we describe Rhodobacter sphaeroides ethylmalonyl-CoA mutase (Ecm), a novel member of the family of coenzyme B12-dependent acyl-CoA mutases, that operates in the recently discovered ethylmalonyl-CoA pathway for acetate assimilation. Ecm is involved in the central reaction sequence of this novel pathway and catalyzes the transformation of ethylmalonyl-CoA to methylsuccinyl-CoA in combination with a second enzyme that was further identified as promiscuous ethylmalonyl-CoA/methylmalonyl-CoA epimerase. In contrast to the epimerase, Ecm is highly specific for its substrate, ethylmalonyl-CoA, and accepts methylmalonyl-CoA only at 0.2% relative activity. Sequence analysis revealed that Ecm is distinct from (2R)-methylmalonyl-CoA mutase as well as isobutyryl-CoA mutase and defines a new subfamily of coenzyme B12-dependent acyl-CoA mutases. In combination with molecular modeling, two signature sequences were identified that presumably contribute to the substrate specificity of these enzymes.

The citric acid cycle has a dual role in central carbon metabolism. On the one hand, it is the terminal respiratory pathway and is used for the complete oxidation of acetyl-CoA to CO 2 . On the other hand, the citric acid cycle is the source of carbon precursors for the synthesis of cell constituents. However, net synthesis of carbon precursors via the citric acid cycle is not possible. Intermediates that are drained from the cycle must be replenished using anaplerotic reaction sequences (1). Therefore, growth on substrates that enter the central carbon metabolism on the level of acetyl-CoA, such as fatty acids, alcohols, and esters but also waxes, alkenes, or C 1 compounds, requires an additional pathway for the assimilation of acetyl-CoA. The glyoxylate cycle that is used for the net conversion of two acetyl-CoA molecules to malate provides a solution for this problem and has been the only known route for 50 years (2).
Recently we proposed the so-called ethylmalonyl-CoA pathway for acetyl-CoA assimilation by Rhodobacter sphaeroides, which lacks isocitrate lyase, the key enzyme of the glyoxylate cycle (3,4). The new pathway converts three molecules of acetyl-CoA, one molecule of CO 2 , and one molecule of bicarbonate to the citric acid cycle intermediates malate and succinyl-CoA (see Fig. 1). Initially two molecules of acetyl-CoA are converted into crotonyl-CoA involving steps that are common to polyhydroxybutyrate synthesis. Crotonyl-CoA is further transformed in a unique reaction sequence to ␤-methylmalyl-CoA that is cleaved in the latter part of the pathway to glyoxylate and propionyl-CoA. Glyoxylate condenses with another molecule of acetyl-CoA to form L-malyl-CoA that is in turn hydrolyzed by a so far unknown thioesterase to malate. Propionyl-CoA is assimilated by carboxylation followed by a carbon skeleton rearrangement step catalyzed by coenzyme B 12 -dependent (2R)-methylmalonyl-CoA mutase yielding succinyl-CoA. This novel assimilation pathway is not only limited to R. sphaeroides but seems to operate in a number of other bacteria that have been reported to lack a functional glyoxylate cycle, like Methylobacterium extorquens and Streptomyces coelicolor (3,4,6,7).
The central and characteristic part of the ethylmalonyl-CoA pathway, the conversion of the C 4 compound crotonyl-CoA to the C 5 compound ␤-methylmalyl-CoA, is not fully understood. The first step in this reaction sequence is a reductive carboxylation of crotonyl-CoA to ethylmalonyl-CoA, an unusual reaction catalyzed by crotonyl-CoA carboxylase/reductase (Ccr) 2 (4). This study aimed at elucidating the steps following the formation of ethylmalonyl-CoA. Here we report the identification and characterization of two enzymes, ethylmalonyl-CoA epimerase and ethylmalonyl-CoA mutase, that are involved in the carbon rearrangement of ethylmalonyl-CoA to methylsuccinyl-CoA (Fig. 1). Although the epimerase is a promiscuous enzyme acting equally well on ethylmalonyl-CoA and methylmalonyl-CoA, ethylmalonyl-CoA mutase is highly specific for its substrate and distinct from the well studied (2R)-methylmalonyl-CoA mutase. Sequence and structural model comparisons of ethylmalonyl-CoA mutase with known methylmalonyl-CoA mutases and isobutyryl-CoA mutase from Streptomyces cinnamonensis revealed interesting structural and functional aspects with implications for the substrate specificities of these coenzyme B 12dependent acyl-CoA mutases.

Bacterial Strains and Growth Conditions
R. sphaeroides strain 2.4.1 (DSMZ 158) was grown at pH 6.7 and 30°C aerobically in the dark or in 2-liter bottles anaerobically in the light (3,000 lux) on defined media supplemented with a 10 mM concentration of the appropriate carbon source as described previously (3). Growth was followed by determining the absorbance at 578 nm (A 578 ), and cells were harvested in midexponential phase at an optical density of 0.5-1.0. For growth studies, R. sphaeroides mutant ecm::kan and wild type were pregrown anaerobically in 100 ml of minimal medium containing 10 mM sodium succinate, and 0.1 ml was transferred to stoppered screw-capped (Hungate) tubes with 10 ml of minimal medium and the appropriate carbon source. The mutant was grown in the presence of 20 g ml Ϫ1 kanamycin. Escherichia coli strains DH5␣, FIGURE 1. The ethylmalonyl-CoA pathway for acetyl-CoA assimilation as proposed for the phototrophic bacterium R. sphaeroides. The conversion of the C 4 compound crotonyl-CoA to the C 5 compound ␤-methylmalyl-CoA involves unique enzymes: crotonyl-CoA carboxylase/reductase (4), mesaconyl-CoA hydratase (3,5), and a proposed enzyme catalyzing the oxidation of methylsuccinyl-CoA to mesaconyl-CoA (methylsuccinyl-CoA dehydrogenase). Ethylmalonyl-CoA mutase and epimerase are described in this work and catalyze the conversion of ethylmalonyl-CoA to methylsuccinyl-CoA (highlighted). The epimerase is a promiscuous enzyme and is also involved in the conversion of propionyl-CoA to succinyl-CoA later in the pathway. BL21(DE3), Rosetta 2(DE3), and S17-1/pir were grown in Luria-Bertani (LB) broth. For conjugation experiments R. sphaeroides was also grown aerobically on LB medium in the dark.

Syntheses
Crotonyl-CoA was synthesized from its anhydride (8). Acrylyl-CoA was synthesized from the free acid by the method of Stadtman (9). CoA-esters were quantified by determining the absorption at 260 nm (⑀ ϭ 22,000 M Ϫ1 cm Ϫ1 ) (10), and the purity was analyzed by a previously described HPLC method (4).

Mutant Construction and Strain Isolation
Chromosomal DNA from R. sphaeroides was isolated using standard techniques. A fragment containing 500 -600 nucleotides flanking regions on either site of the PstI sites within the ecm gene was amplified using the forward primer, 5Ј-ATG GTA AGC TTC CTC TAG AA TCT GTC TGC TGC AAC-3Ј, to introduce HindIII and XbaI sites (underlined) and the reverse primer, 5Ј-TCC GGT AC CAG TCT AGA TAT GGG CCA GCT CTT C-3Ј, to introduce KpnI and XbaI sites (underlined). PCR was performed using Pfu polymerase (Genaxxon Bioscience GmbH) for 25 cycles, including denaturation for 1 min, annealing at 62°C for 1 min, and extension at 72°C for 5 min. The PCR product was isolated and cloned into pUC19 to obtain plasmid pAS10. A kanamycin resistance cassette (kan) was amplified using forward primer 5Ј-AAC TGC AGT TAG  AAA AAC TCA TCG AGC-3Ј, reverse primer 5Ј-TTC TGC  AGA AAG CCA CGT TGT GTC TCA AAA TC-3Ј, and pUC4KSAC (Promega) as a template. The 159-nucleotide PstI fragment within the ecm gene was replaced by the kanamycin resistance cassette, resulting in plasmid pASK101. The plasmid was digested with XbaI, and the fragment containing the interrupted ecm gene was ligated into pJQ200mp18 (11), resulting in plasmid pJS8meaAKJ. This plasmid was transferred into R. sphaeroides by conjugation with E. coli S17-1/pir carrying pJS8meaAKJ. Donor and recipient strains were grown on LB medium to midexponential phase (A 578 nm ϭ 0.4 -0.5) with 20 g ml Ϫ1 kanamycin and 15 g ml Ϫ1 gentamycin in the case of the donor strain. Cells (from a 15-ml culture) were collected by centrifugation at 6,000 ϫ g for 10 min, washed with LB medium, mixed in a 1:1 ratio, and centrifuged again. The pellet was resuspended in 200 l of LB medium and placed as a single drop on an LB agar plate for mating. After incubation at 30°C in the dark for 24 h, the cells were suspended in 200 l of LB medium, diluted from 10 Ϫ1 to 10 Ϫ6 , and plated on LB agar with 15 g ml Ϫ1 kanamycin (positive selection for crossover event) and 10% sucrose (negative selection for single crossover and donor). Conjugants were picked after 4 -6 days of incubation (30°C anaerobically in the light). The deletion/insertion mutation of the ecm gene was verified by PCR analyses.

Cell Extracts and Qualitative Enzymatic Measurements
Cell extracts of R. sphaeroides were prepared by suspending 0.3-0.5 g of cells in 0.5 ml of 20 mM Tris⅐HCl (pH 7.8) containing 50 g ml Ϫ1 DNase I. Glass beads (1.1 g, 0.1-0.25-mm diameter) were added, and the suspension was treated for 9 min at 30 Hz and 4°C in a mixer mill (Retsch, Haare, Germany). After centrifugation (10 min, 20,000 ϫ g, 4°C), the supernatant (cell extract, 5-10 mg ml Ϫ1 protein) was tested for ethylmalonyl-CoA or methylmalonyl-CoA transforming activity. Protein concentrations were determined by the method of Bradford using bovine serum albumin as the standard (12).
Ethylmalonyl-CoA Mutase Assay-Radioactive labeled ethylmalonyl-CoA was synthesized in situ from crotonyl-CoA, H 14 CO 3 Ϫ , NADPH, and recombinant Ccr and subsequently used as substrate: the "substrate mixture" (3.07 ml) contained 76 mM Tris⅐HCl (pH 7.8), 4.9 mM NADPH, 2.0 mM crotonyl-CoA, 17.7 mM NaHCO 3 , 0.8 MBq ml Ϫ1 NaH 14 CO 3 , and 120 g of recombinant Ccr. After 10 -30 min of incubation at 30°C, 90 l of the substrate mixture were added to 20 l of cell extract protein that had been preincubated for 10 min at 30°C in the presence of 0.025 mol of coenzyme B 12 . An aliquot of 30-l samples was taken at different time points and stopped with 5 l of 4 M KOH to follow the fate of ethylmalonyl-CoA. The samples were incubated for 20 min at 80°C to hydrolyze the CoA-esters and then acidified with 10 l of H 2 SO 4 . Separation of the free acids was performed with thin layer chromatography on silica gel 60 F 254 plates (Merck) with CHCl 3 :acetic acid (5:1, v/v) as solvent and subsequent detection of radioactivity by phosphorimaging. The dependence on coenzyme B 12 was examined using the supernatant of a 35% ammonium sulfate precipitation of cell extract protein that had been dialyzed overnight against 20 mM Tris⅐HCl and preincubated for 10 min in the presence (0.025 mol) or absence of coenzyme B 12 .

Product Isolation and Identification by NMR
Cell extract of R. sphaeroides was prepared by suspending 3 g of cells in 6 ml of 20 mM Tris⅐HCl (pH 7.8) containing 0.1 mg l Ϫ1 DNase I. The suspension was passed twice through a French pressure cell at 137 megapascals and 4°C, and the cell lysate was centrifuged (100,000 ϫ g, 4°C, 1 h). Coenzyme B 12 (0.635 mol) was added to 1.6 ml of the supernatant (cell extract protein, approximately 60 mg ml Ϫ1 ) and incubated at 30°C. 3-Carboxy-13/14 C-labeled ethylmalonyl-CoA was produced in a substrate mixture (15.7 ml) that contained 79 mM Tris⅐HCl (pH 7.8), 5.1 mM NADPH, 2.0 mM crotonyl-CoA, 31.8 mM NaH 13 CO 3 , 29 kBq ml Ϫ1 NaH 14 CO 3 , and 6 mg of recombinant Ccr. After 12 min of incubation at 30°C, the substrate mixture was combined with the B 12 -incubated cell extract. After incubation for 90 min at 30°C, 1.6 ml of 4 M KOH were added, and the mixture was incubated for 30 min at 80°C to hydrolyze the CoA ester into free acids. Protein was removed by adding 25% HCl to pH ϭ 0 followed by a centrifugation step (38,000 ϫ g, 4°C, 20 min). The free acids were extracted from the supernatant by solvent extraction with ethyl acetate (four times, 1:1, v/v), and the organic phase was concentrated to a volume of 1.5 ml by evaporation. The acids were separated by preparative thin layer chromatography on silica gel 60 F 254 plates (Merck) with CHCl 3 :acetic acid (5:1, v/v) as solvent and subsequent detection of radioactivity. The radioactive labeled compound co-eluting with methylsuccinate was scratched from the plate. The material was extracted five times with 10 ml of methanol. The organic solvent was concentrated by evaporation and lyophilized overnight, and the remainder was dissolved in MeOH-d 4 for NMR spectroscopy. NMR spectra were recorded with a Bruker Avance DRX-400 spectrometer at 27°C. Chemical shifts were recorded and reported in ppm relative to MeOH-d 4 ( 1 H: ␦ ϭ 3.31, 13 C: ␦ ϭ 49.15) as internal standard.

Cloning and Heterologous Expression of ecm and epi from R. sphaeroides in E. coli
The gene encoding ethylmalonyl-CoA mutase (ecm) was amplified from R. sphaeroides chromosomal DNA by using the forward primer 5Ј-CCT TCA TAT GAC CCA GAA GGA TAG CCC CTG-3Ј introducing an NdeI site (underlined) and the reverse primer 5Ј-CGG GAA GCT TGG GAT CCT ATT CCG CC-3Ј introducing a HindIII site (underlined). PCR was performed with PfuTurbo polymerase (Stratagene, Cedar Creek, TX) for 33 cycles, including denaturation for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 2.5 min. The PCR product was isolated and cloned into pET16b (Invitrogen) to obtain pTE33 for expression of ecm and production of an N-terminal deca-His tag fusion protein. The gene encoding ethylmalonyl-CoA/methylmalonyl-CoA epimerase (epi) was amplified with forward primer 5Ј-GGA GAG CAT ATG ATC GGA CGC CTG AAC CAT GTG G-3Ј introducing a NdeI site (underlined) and reverse primer 5Ј-GCC GGT GAA GCT TAT ACC TGC TCG AGC TCC ACG-3Ј introducing a HindIII site (underlined) and a PCR program of 34 cycles with denaturation for 45 s, annealing at 60°C for 45 s, and extension at 72°C for 80 s. The PCR product was cloned into pET16b resulting in pTE45 to express an N-terminal deca-His-tagged version of Epi. Competent E. coli BL21(DE3) or Rosetta 2 (DE3) were transformed with the appropriate plasmid and grown at 37°C in LB medium with 100 g ml Ϫ1 ampicillin. Expression was induced at A 578 ϭ 0.6 -0.9 with 0.5 mM isopropyl thiogalactopyranoside, and the temperature was lowered to 30°C. For expression of ecm, 1 ml of Pfennig vitamin solution VL-7 (13) was added per liter of LB medium. Cells were harvested after additional growth for 3 h and stored at Ϫ80°C until use. Heterologous production of Ccr in a 200-liter scale and purification of the recombinant protein have been described previously (4).

Purification of Recombinant His-tagged Ecm and Epi
All purification steps were performed at 4°C. Frozen cells were suspended in a double volume of 20 mM Tris⅐HCl (pH 7.8) containing 0.1 mg l Ϫ1 DNase I. The suspension was passed twice through a chilled French pressure cell at 137 megapascals, and the cell lysate was centrifuged (100,000 ϫ g) at 4°C for 1 h. An aliquot of the supernatant (2-5 ml, 70 -100 mg of protein) was applied at a flow rate of 1 ml min Ϫ1 onto a 1-ml nickel-Sepharose Fast Flow column (HisTrap FF; Amersham Biosciences) that had been equilibrated with buffer A (20 mM Tris⅐HCl and 200 mM KCl (pH 7.8)). After application of cell extract, the column was washed with buffer A and buffer A containing 75 mM imidazole at a flow rate of 1 ml min Ϫ1 to elute unwanted protein. Recombinant Ecm or Epi was eluted with buffer A containing 500 mM imidazole. The enzymes were desalted and concentrated by ultrafiltration with an Amicon XM 50 (Ecm) or an Amicon YM 10 (Epi) membrane (Millipore, Eschborn, Germany). The protein (8 -10 mg) was stored at Ϫ20°C in 10 mM Tris⅐HCl (pH 7.8) with 50% glycerol. Recombinant Ccr was purified from cell extracts as described before (4).

Quantitative Enzymatic Measurements of Recombinant Enzymes
Ecm and Epi were measured quantitatively by an HPLCbased assay.
Ethylmalonyl-CoA Mutase-Radioactive labeled ethylmalonyl-CoA was synthesized from crotonyl-CoA, H 14 CO 3 Ϫ , and recombinant Ccr and subsequently used as substrate. The substrate mixture (1.07 ml) contained 80 mM Tris⅐HCl (pH 7.8), 3.7 mM NADPH, 1.9 mM crotonyl-CoA, 7.9 mM NaHCO 3 , 0.4 MBq ml Ϫ1 NaH 14 CO 3 , and 230 g of recombinant Ccr. After incubation at 30°C for 10 min, crotonyl-CoA was completely transformed to ethylmalonyl-CoA as shown by HPLC, and the reaction was stopped by boiling the reaction mixture for 5 min. To start the ethylmalonyl-CoA mutase assay, 212 l of the substrate mixture were added to 20 l of a "protein solution" containing 0.12 mol of coenzyme B 12 , an excess of partially purified methylmalonyl-CoA racemase fraction from Propionibacterium freudenreichii subsp. shermanii (15), and 3-6 g of recombinant Ecm. The reaction was stopped at different time points by removing 50-l samples and adding 5 l of 20% formic acid; acidification also avoids hydrolysis of the unstable methylsuccinyl-CoA. The samples were diluted with 50 l of water and centrifuged (15 min, 20,000 ϫ g) to remove denatured protein. The supernatant was analyzed by reversed-phase HPLC on a C 18 column (LiChrospher 100, end-capped, 5 m, 125 ϫ 4 mm; Merck). The column was developed for 7 min under isocratic conditions with 100 mM NaH 2 PO 4 (pH 4.0) in 7.5% methanol (v/v) followed by a linear 10-min gradient from 0 to 60% 100 mM sodium acetate (pH 4.2) in 90% methanol (v/v) at a flow rate of 1 ml min Ϫ1 . Reaction products and standard compounds were detected by UV absorbance with a Waters 996 photodiode array detector. Radioactivity of eluting compounds was also monitored by a Ramona 2000 radioactive monitor (Raytest, Straubenhardt, Germany) connected in series. Retention times were 11.7 min for methylmalonyl-CoA, 12.3 min for succinyl-CoA, 12.5 min for ethylmalonyl-CoA, and 13.1 min for meth-ylsuccinyl-CoA (radioactive monitoring). The amount of product formed was calculated from the relative peak area. The apparent K m value of ethylmalonyl-CoA was determined by varying the end concentration of ethylmalonyl-CoA from 0.12 to 1.8 mM while keeping coenzyme B 12 at a saturating concentration (506 M). The apparent K d of coenzyme B 12 was determined by varying the end concentration of coenzyme B 12 from 0.25 to 506 M at a saturating ethylmalonyl-CoA concentration (1.8 mM). The pH optimum was determined using 16 mM Tris⅐HCl (pH 7.8) instead of 80 mM in the substrate mixture. The pH was subsequently adjusted by addition of 92 l of the substrate mixture to 55 l of a protein solution containing 0.13 mol of coenzyme B 12 , excess amounts of methylmalonyl-CoA racemase from P. freudenreichii subsp. shermanii, 3 g of recombinant Ecm, and an Ellis and Morrison (16)  Ethylmalonyl-CoA Epimerase-To measure ethylmalonyl-CoA epimerase activity, recombinant Ecm was used in excess, and the amount of Epi was kept rate-limiting. Also the substrate mixture for the synthesis of ethylmalonyl-CoA (from crotonyl-CoA) was not stopped by boiling to avoid spontaneous epimerization of ethylmalonyl-CoA. Instead 212 l of the substrate mixture were added directly to 38 l of a protein solution containing 0.13 mol of coenzyme B 12 , 0.20 mol of CoCl 2 , 0.08 -0.24 g of recombinant Epi, and 32-64 g of recombinant Ecm. The apparent K m value of ethylmalonyl-CoA was examined by varying the end concentration of ethylmalonyl-CoA (0.06 -0.88 mM) in the assay at a saturating coenzyme B 12 concentration (506 M). Additionally NaH 14 CO 3 was increased to 3.2 MBq ml Ϫ1 for the synthesis of ethylmalonyl-CoA by Ccr. To test the influence of divalent cations, EDTA was added to a 1.3 mg ml Ϫ1 Epi solution to a final concentration of 2 mM and incubated overnight. An aliquot of this solution containing 0.03-0.3 g of Epi was diluted into protein solution (final volume, 38 l) containing 0.2 mol of CoCl 2 , MgCl 2 , MnCl 2 , or NiCl 2 or no divalent cation before the reaction was started by adding 212 l of substrate mixture.
Methylmalonyl-CoA Epimerase-Methylmalonyl-CoA epimerizing activity of recombinant Epi was measured analogously to ethylmalonyl-CoA epimerizing activity. Crotonyl-CoA was replaced by acrylyl-CoA for substrate synthesis resulting in the formation of radioactively labeled methylmalonyl-CoA. Recombinant methylmalonyl-CoA mutase 3 was used in excess, and the amount of Epi (10 -40 ng) was kept ratelimiting. The apparent K m value of methylmalonyl-CoA was examined by varying the concentration of methylmalonyl-CoA from 0.06 to 0.88 mM while keeping coenzyme B 12 at saturating concentrations (506 M).

Phylogenetic Analysis
The genomic BLAST interface (February 13, 2008) at the National Center for Biotechnology Information (NCBI) was used to search 592 fully sequenced genomes with the amino acid sequences of methylmalonyl-CoA mutase (large subunit) from P. freudenreichii subsp. shermanii (NCBI accession number P11653.3), isobutyryl-CoA mutase (large subunit) from S. cinnamonensis (NCBI accession number AAC08713.1), and ethylmalonyl-CoA mutase from R. sphaeroides (NCBI accession number YP_354045). All hits with an expectation value Ͼe Ϫ20 were controlled for the presence of a substrate binding and a coenzyme B 12 binding domain. In absence of either domain, the genomic context was analyzed for the presence of an open reading frame encoding the missing subunit. When both subunits were present, their sequences were linked to a concatamer. All hits where the substrate binding domain was fused to an argK-like domain were excluded because these proteins are not characterized yet and their function as bona fide mutases remains to be shown. The amino acid sequences were aligned using ClustalW as implemented in the BioEdit 7.0.9.0. software package, and the sequences obtained were used for phylogenetic analysis. Phylogenetic trees were constructed using neighbor-joining algorithms as implemented in the Tree-ConW 1.3b software package or maximum parsimony and distance matrix algorithms as implemented in the PHYLIP 3.67 software package.

Computational Modeling
Ethylmalonyl-CoA mutase was modeled using the SWISS-MODEL Automated Comparative Protein Modeling Server (November 6, 2007) with the structure of the methylmalonyl-CoA mutase substrate complex from P. freudenreichii subsp. shermanii (Protein Data Bank code 4REQ) as template. The software packages of DeepView 3.7 and University of California San Francisco Chimera 1 were used to visualize and overlay the structure models.

Coenzyme B 12 -dependent Conversion of Ethylmalonyl-CoA to
Methylsuccinyl-CoA in Cell Extracts of R. sphaeroides-To follow the fate of ethylmalonyl-CoA in cell extracts of R. sphaeroides, 3-carboxy-14 C-labeled ethylmalonyl-CoA was produced enzymatically from crotonyl-CoA, H 14 CO 3 Ϫ , and NADPH by Ccr from R. sphaeroides and used in subsequent experiments. Cell extracts of acetate-grown R. sphaeroides were able to convert this [3-carboxy-14 C]ethylmalonyl-CoA into a radioactive labeled product, and the conversion was dependent on the presence of coenzyme B 12 . After hydrolysis of CoA-thioesters, the product of the conversion co-eluted with methylsuccinate on thin layer chromatography plates (Fig. 2) and was isolated from preparative scale using 3-carboxy-13 Clabeled ethylmalonyl-CoA. One-dimensional ( 1 H, 13 C) and two-dimensional (correlation spectroscopy, heteronuclear multiple bond correlation) NMR experiments confirmed the product as [4-carboxy- 13 C]methylsuccinate, suggesting that a rearrangement of the carbon skeleton had taken place (supplemental Fig. S1). We conclude that a coenzyme B 12 -dependent ethylmalonyl-CoA mutase catalyzed this reaction in cell extracts of R. sphaeroides.
Insertional Inactivation of ecm, the Putative Ethylmalonyl-CoA Mutase Gene, and Characterization of the Mutant ecm::kan-Sequence analysis identified two genes with coenzyme B 12 binding motifs encoding putative mutases in the complete genome of R. sphaeroides (GenBank TM accession numbers CP000143 and CP000144). One gene (mcm) was located downstream of genes annotated to encode both subunits of propionyl-CoA carboxylase. The corresponding protein (Mcm, NCBI accession number ABA78347) had 61% sequence identity to the well characterized methylmalonyl-CoA mutase from P. freudenreichii subsp. shermanii (17)(18)(19)(20) and was shown to catalyze the rearrangement of methylmalonyl-CoA, the product of propionyl-CoA carboxylation, to succinyl-CoA. 3 The other gene (NCBI accession number ABA80144) clustered with ccr and showed 66% amino acid sequence identity to meaA from M. extorquens. meaA was able to rescue a chemical-induced methanol and ethanol assimilation-deficient mutant of M. extorquens (21)(22)(23)(24), and mutation of a homologous gene in Streptomyces collinus also abolished the assimilation of acetyl-CoA (7). The function of this enzyme in acetyl-CoA assimilation and its physiological substrate, however, is not known. To investigate its function in the ethylmalonyl-CoA pathway of R. sphaeroides, this gene, called ecm (for ethylmalonyl-CoA mutase), was inactivated by homologous recombination. Thereby part of ecm was replaced by kan, resulting in a mutant called ecm::kan that was tested subsequently for growth on different carbon sources.
R. sphaeroides ecm::kan was able to grow with carbon substrates that did not require the operation of the (complete) eth-ylmalonyl-CoA pathway (succinate, propionate/HCO 3 Ϫ , or acetate plus glyoxylate) but was unable to use acetate or acetoacetate as the sole carbon source (Fig. 3). The latter compounds rely on the central reactions of ethylmalonyl-CoA ( Fig.  1), indicating that ecm is involved on the level of C 5 compounds and further supporting the hypothesis that the protein might act as an ethylmalonyl-CoA mutase. The fact that growth on propionate/HCO 3 Ϫ , which is known to involve methylmalonyl-CoA mutase, was not affected by the mutant ecm::kan suggested that two distinct coenzyme B 12 -dependent mutases are operating in the ethylmalonyl-CoA pathway.
Ethylmalonyl-CoA and Methylmalonyl-CoA Mutase Are Two Distinct Enzymes-To test for the presence of two distinct coenzyme B 12 -depending mutases, the rearrangement of ethylmalonyl-CoA to methylsuccinyl-CoA and of methylmalonyl-CoA to succinyl-CoA was measured in cell extracts of R. sphaeroides wild type and mutant ecm::kan. Because Ccr accepts both crotonyl-CoA and acrylyl-CoA as substrates, 3 both 3-carboxy-14 C-labeled ethylmalonyl-CoA and 3-carboxy-14 C-labeled methylmalonyl-CoA were synthesized in vitro by Ccr and were subsequently used for cell extract experiments. Whereas cell extracts of wild-type R. sphaeroides grown with acetate plus glyoxylate showed ethylmalonyl-CoA and methylmalonyl-CoA converting activities, ethylmalonyl-CoA converting activity was undetectable in cell extracts of the mutant ecm::kan grown under the same conditions (Fig. 4). Yet methylmalonyl-CoA was still converted to succinyl-CoA by cell extracts of the ecm::kan mutant (Fig. 4B) consistent with reports on the presence of methylmalonyl-CoA mutase activity in cell extracts of meaA mutants of M. extorquens and S. collinus (7,21,24). This clearly demonstrated that ethylmalonyl-CoA mutase is distinct from methylmalonyl-CoA mutase.
Expression of ecm and Purification of Ethylmalonyl-CoA Mutase-To study its biochemical properties, ecm was heterologously expressed in E. coli, and the N-terminal His-tagged protein was purified from crude extracts in an one affinity chromatographic step. Denaturing gel electrophoresis revealed the presence of a major protein band (75 kDa; supplemental  Ϫ (t(0)). The reaction was started by cell extract protein of acetate-grown R. sphaeroides (supernatant of a 35% ammonium sulfate fraction dialyzed overnight) in the presence (ϩcoenzyme B 12 ) or absence (Ϫcoenzyme B 12 ) of coenzyme B 12 . Samples were taken after 90 min and stopped with KOH to hydrolyze the corresponding coenzyme A-thioesters. Free acids were separated by thin layer chromatography, and radioactivity was detected. The positions of non-radioactive labeled authentic ethylmalonate (EM) and methylsuccinate (MS) were detected by bromcresol green solution and are marked. peptide mass fingerprint analysis, the major band corresponded to full-length Ecm, whereas the two weak bands were C-terminally truncated versions of Ecm lacking the B 12 binding domain and different parts of a linker region.
Ethylmalonyl-CoA Mutase Depends on an Epimerase-like Protein-Purified recombinant Ecm was unable to form methylsuccinyl-CoA from ethylmalonyl-CoA when ethylmalonyl-CoA was produced by reductive carboxylation of crotonyl-CoA and recombinant Ccr. However, addition of cell extract of R. sphaeroides ecm::kan mutant to the enzyme assay restored ethylmalonyl-CoA mutase activity (Fig. 5A). This indicated that the enzyme itself is competent but that an additional factor present in cell extract is required for Ecm to be active. Note that ethylmalonyl-CoA exists in two stereoisomeric forms, one of which is probably selectively formed by Ccr. Hence the situation might be similar to methylmalonyl-CoA mutase that is dependent on methylmalonyl-CoA epimerase to convert (2S)methylmalonyl-CoA, the product of propionyl-CoA carboxylation, to (2R)-methylmalonyl-CoA, the substrate for Mcm (25,26). We tested the ability of methylmalonyl-CoA epimerase to accept ethylmalonyl-CoA as a substrate and to complement the activity of Ecm. Indeed addition of a methylmalonyl-CoA epimerase fraction from P. freudenreichii subsp. shermanii to Ecm restored ethylmalonyl-CoA converting activity (Fig. 5B), suggesting the involvement of a similar enzymatic activity in the ethylmalonyl-CoA pathway.
Identification, Molecular Expression, and Characterization of Ethylmalonyl-CoA/Methylmalonyl-CoA Epimerase-A homology search in the genome of R. sphaeroides using the amino acid sequence from methylmalonyl-CoA epimerase of P. freudenreichii subsp. shermanii (NCBI accession number AAL57846) identified only one possible candidate annotated to encode glyoxalase I (NCBI accession number ABA79990), an enzyme of structural and functional homology to methylmalonyl-CoA epimerase (27). The gene called epi (for epimerase) was cloned and expressed, and the protein was heterologously produced as a histidine fusion protein in E. coli (supplemental Fig. S2B). The purified protein was able to substitute Propionibacterium epimerase in the overall conversion of crotonyl-CoA/HCO 3 Ϫ to methylsuccinyl-CoA using recombinant Ccr and Ecm (data not shown), indicating that Epi acts as an ethylmalonyl-CoA epimerase for R. sphaeroides.
A coupled assay was developed to determine kinetic parameters of Epi. Ethylmalonyl-CoA was synthesized from crotonyl-CoA/H 14 CO 3 Ϫ by Ccr and used as substrate with excess amounts of Ecm and rate-limiting amounts of Epi (Fig. 6A). The dependence of Epi for ethylmalonyl-CoA followed Michaelis-Menten kinetics with an apparent K m of 40 M and v max of 110 units mg Ϫ1 . To test whether Epi also catalyzes the epimerization of methylmalonyl-CoA, the assay was modified. Methylmalonyl-CoA was synthesized from acrylyl-CoA/H 14 CO 3 Ϫ using recombinant Ccr, and Ecm was replaced with excess recombinant methylmalonyl-CoA mutase encoded by mcm from R. sphaeroides 3 (Fig. 6B). Interestingly with a v max of 120 units mg Ϫ1 and an apparent K m of 80 M, the kinetic parameters for Epi toward methylmalonyl-CoA are comparable to its kinetic properties with ethylmalonyl-CoA, suggesting that Epi is a promiscuous enzyme catalyzing both isomerization reactions in vivo. The activities of methylmalonyl-CoA epimerases from different sources have been reported to be stimulated by Co 2ϩ (28,29). Incubation of Epi for 20 min with Co 2ϩ (0.4 mM) increased enzymatic activity 4-fold (440 units mg Ϫ1 ), whereas incubation of Epi with 2 mM EDTA overnight led to a complete loss of activity. The dependence on divalent cations was studied by incubation of EDTA-inactivated Epi with different metal  ions. Co 2ϩ and Mn 2ϩ restored full activity (440 units mg Ϫ1 ), whereas Mg 2ϩ and Ni 2ϩ were not able to activate the enzyme. Residues that have been implied for Co 2ϩ binding in methylmalonyl-CoA epimerase from P. shermanii are conserved in the enzyme from R. sphaeroides except Gln-65 is replaced by a glutamate residue (27).
Molecular Properties of Recombinant Ethylmalonyl-CoA Mutase-The newly developed coupled assay (see above) also enabled the kinetic characterization of Ecm using excess amounts of Epi and rate-limiting amounts of Ecm. The specific activity of ethylmalonyl-CoA mutase in cells of R. sphaeroides grown on acetate was 50 nmol min Ϫ1 mg Ϫ1 of protein. The purified recombinant ethylmalonyl-CoA mutase followed Michaelis-Menten kinetics with apparent K m values of 60 M for ethylmalonyl-CoA. Half-maximal activity for Ecm toward coenzyme B 12 was 2 M. This value can be taken as the dissociation constant because Ecm is only active with coenzyme B 12 bound. The maximal specific activity (v max ) was determined as 7 units mg Ϫ1 , a value that represents a lower limit because truncated and therefore inactive protein is present in the purified enzyme fraction (supplemental Fig. S2A). The enzyme was tested in a pH range of 6.0 -10.0 (0.5 pH units/step) and had a broad pH optimum around 6.5-8.0. The calculated molecular mass of Ecm is 74 kDa, and gel filtration chromatography of the native enzyme gave an apparent molecular mass of 153 kDa, suggesting an apparently homodimeric subunit structure (␣ 2 ). Ethylmalonyl-CoA mutase accepted methylmalonyl-CoA as substrate with only 0.2% relative activity.
Identification of Ethylmalonyl-CoA Mutase Defines a New Subfamily of B 12 -dependent Acyl-CoA Mutases-The sequence distance relationship of known acyl-CoA mutases with ethylmalonyl-CoA mutase from R. sphaeroides and closely related proteins was studied. For this analyses, sequences of biochemically characterized (homo-and heterodimeric) methylmalonyl-CoA, isobutyryl-CoA, and 2-hydroxybutyryl-CoA mutases (17, 18, 30 -36) or corresponding acyl-CoA mutases from fully sequenced genomes were used in which the physiological role has been reasonably suggested from the genomic context or experimental studies. In the case of heterodimeric methylmalonyl-CoA mutases that are composed of an active ␣ and an inactive ␤ subunit, only the sequences of the catalytically competent ␣ subunit were included in the analysis.
Interestingly all coenzyme B 12 -dependent acyl-CoA mutases were clearly clustered according to their function/substrate specificity and not to the proposed phylogenetic positioning of the corresponding species (Fig. 7A; for a more detailed analysis see supplemental Fig. S3). The only exceptions are archaeal methylmalonyl-CoA mutases that appear more closely related to isobutyryl-CoA mutases than to bacterial and eukaryotic/ mitochondrial methylmalonyl-CoA mutases.
According to this functional clustering, ethylmalonyl-CoA mutase from R. sphaeroides formed a distinct subclade with proteins encoded by the genomes of various ␣-proteobacteria and actinomycetes (Fig. 7B), including MeaA from M. extorquens and Streptomyces sp. (7,21,24). In each case, the corresponding gene clustered with a gene likely to encode ccr on the chromosome, and all other genes involved in the ethylmalonyl-CoA pathway were also present in the genomes of these organisms (data not shown). We concluded that all proteins of this subclade are ethylmalonyl-CoA mutases that function in the ethylmalonyl-CoA pathway of the respective bacteria.
Signature Sequences of Ethylmalonyl-CoA Mutase May Have Mechanistic Implications-The identification of the ethylmalonyl-CoA mutase subfamily provided the opportunity to study the molecular basis of the very narrow substrate specificity of acyl-CoA dependent mutases. Primary structure comparisons of methylmalonyl-CoA-and ethylmalonyl-CoA mutases identified two signature sequences that were unique to either Radioactively labeled ethylmalonyl-CoA was synthesized using recombinant Ccr and crotonyl-CoA. The assay was started by addition of ethylmalonyl-CoA mutase and ethylmalonyl-CoA/methylmalonyl-CoA epimerase (ϩepi). In a control experiment, ethylmalonyl-CoA/methylmalonyl-CoA epimerase was omitted from the assay (Ϫepi). Samples were withdrawn from the assay after 1 min and analyzed by reversed-phase HPLC. The retention times were 2.7 min for H 14 CO 3 Ϫ , 12.5 min for ethylmalonyl-CoA, and 13.1 min for methylsuccinyl-CoA. The activity of ethylmalonyl-CoA/methylmalonyl-CoA epimerase was quantified from the amount of product formed at several time points. B, methylmalonyl-CoA epimerase activity. Radioactively labeled methylmalonyl-CoA was synthesized using recombinant Ccr and acrylyl-CoA. The assay was started by addition of methylmalonyl-CoA mutase and ethylmalonyl-CoA/methylmalonyl-CoA epimerase (ϩepi). In a control experiment, ethylmalonyl-CoA/methylmalonyl-CoA epimerase was omitted (Ϫepi). Samples were taken after 1 (Ϫepi) or 5 min (ϩepi) and analyzed by reversed-phase HPLC. The retention times were 11.7 min for methylmalonyl-CoA and 12.3 min for succinyl-CoA. enzyme and located in the central part of the proteins (supplemental Fig. S4). Signature sequence I displayed a conserved exchange of a histidine in Mcm to a glycine in Ecm, whereas an additional stretch of three amino acids was observed in signature sequence II, indicating a possible correlation of amino acid sequence/structure and substrate specificity.
These results were supported by homology modeling of Ecm with the structure of a methylmalonyl-CoA mutase succinyl-CoA complex of P. freudenreichii subsp. shermanii as template (Protein Data Bank code 4REQ; sequence identity, 61%). In this model, both signature sequences were localized in direct vicinity to the succinyl moiety bound to the active center (supplemental Fig. S5). Moreover the structure model suggested that the histidine to glycine exchange of signature sequence I contributes considerably to the altered substrate specificity, emphasizing the striking correlation of sequence conservation, three-dimensional structure, and functional context in coenzyme B 12 -dependent acyl-CoA mutases.

Rearrangement of Ethylmalonyl-CoA to Methylsuccinyl-
CoA-The assimilation of C 1 and C 2 compounds via the recently described ethylmalonyl-CoA pathway depends on the conversion of ethylmalonyl-CoA into methylsuccinyl-CoA, a central reaction in the unique reaction sequence from crotonyl-CoA to ␤-methylmalyl-CoA (Fig. 1). This challenging carbon skeleton rearrangement is catalyzed by the combined action of ethylmalonyl-CoA/methylmalonyl-CoA epimerase and ethylmalonyl-CoA mutase, a new coenzyme B 12 -dependent enzyme described in this study.
Ethylmalonyl-CoA/Methylmalonyl-CoA Epimerase: a Promiscuous Enzyme with a Dual Role in the Ethylmalonyl-CoA Pathway-The formation of methylsuccinyl-CoA from ethylmalonyl-CoA in vivo requires not only ethylmalonyl-CoA mutase but also an additional enzyme that could be identified as ethylmalonyl-CoA epimerase. This suggests the following reaction sequence in which crotonyl-CoA is first carboxylated to (2S)-ethylmalonyl-CoA that is then converted by ethylmalonyl-CoA epimerase into its 2R-stereoisomer. (2R)-Ethylmalonyl-CoA in turn serves as bona fide substrate for ethylmalonyl-CoA mutase as proposed from its homology to methylmalonyl-CoA mutase, which has been shown to be specific for (2R)-methylmalonyl-CoA (25,26).
Interestingly ethylmalonyl-CoA epimerase is a promiscuous enzyme accepting methylmalonyl-CoA with comparable kinetic properties as a substrate. This implicates a dual role of ethylmalonyl-CoA/methylmalonyl-CoA epimerase in the ethylmalonyl-CoA pathway because the later conversion of propionyl-CoA to succinyl-CoA is dependent on the action of a methylmalonyl-CoA epimerase (37,38). The identification of ethylmalonyl-CoA/methylmalonyl-CoA epimerase therefore raises the question whether broad substrate specificity is a common feature of this enzyme family. Alternatively the promiscuity observed could reflect a specific feature of those enzymes involved in the ethylmalonyl-CoA pathway.
Ethylmalonyl-CoA Mutase: a New Acyl-CoA Mutase-In contrast to the epimerase, ethylmalonyl-CoA mutase is highly specific for ethylmalonyl-CoA and accepts methylmalonyl-CoA only with very low activity (0.2%). Ethylmalonyl-CoA mutase is therefore distinct from its paralog methylmalonyl-CoA mutase (54% sequence similarity) that is also up-regulated during growth of R. sphaeroides on acetate (3) and catalyzes the conversion of methylmalonyl-CoA to succinyl-CoA later in the pathway. 3 Despite their high sequence similarity, ethylmalonyl-CoA mutase (this study) and methylmalonyl-CoA mutase (19) 3 are highly specific for their respective substrate; this can be explained by the radical reaction mechanism of those enzymes. This mechanism implies the tight binding of the substrate to the active center of the enzyme to exclude water and to prevent any unwanted side reaction. The CoA-ester is therefore bound along a cleft in the substrate binding domain that is associated with a large conformational change to bury the substrate, close up the active site, and initiate the radical reaction as shown for methylmalonyl-CoA mutase (18). Consistent with this model of "negative catalysis" (40,41), alteration of the substrate specificity should then require a substantial change in the active site that otherwise would result in suboptimal binding of the altered substrate and, therefore, in a non-functional enzyme. A first proof of principle was provided by changing a specific active site amino acid in methylmalonyl-CoA mutase to the corresponding residue present in isobutyryl-CoA mutase: the turnover number for methylmalonyl-CoA was decreased by 10 4 (42), and isobutyryl-CoA was accepted by the variant. But instead of becoming a substrate and being converted into butyryl-CoA, isobutyryl-CoA acted as a suicide inhibitor because of irreversible inactivation of the enzyme by uncontrolled electron transfer (42).
Although methylmalonyl-CoA and isobutyryl-CoA are comparable with respect to the C-2 atom where the rearrangement reaction takes place (30), ethylmalonyl-CoA is a more bulky substrate (supplemental Fig. S6). A change in enzyme specificity from methylmalonyl-/isobutyryl-CoA to ethylmalonyl-CoA should therefore require a major change of the active site to tightly bind this bulkier acyl moiety (at the C-2 atom) that would allow a controlled course of the rearrangement reaction. Such an adaptation of ethylmalonyl-CoA mutase to its more spacious substrate may be reflected in both signature sequences that have been identified as part of the active site and are conserved within the subfamily of ethylmalonyl-CoA mutases. Especially the substitution of a conserved histidine (position 328 in Mcm of P. freudenreichii subsp. shermanii) to a less spacious glycine (homologous position 255 in Ecm of R. sphaeroides) in signature sequence I could provide more space for the preferential binding of the additional CH 3 group in the acyl moiety of ethylmalonyl-CoA. The situation for signature sequence II is less clear. Here an additional stretch of three amino acids was observed for ethylmalonyl-CoA mutase, possibly resulting in a structural change in this part of the protein. This insertion is accompanied by the exchange of a conserved asparagine (position 366 in Mcm of P. freudenreichii subsp. shermanii) to a proline (homologous position 296 in Ecm of R. sphaeroides). Prolines are known to have a strong impact on the three-dimensional structure of proteins, emphasizing a possible structural difference between ethylmalonyl-CoA and methylmalonyl-CoA mutase. Considering the similarity of methylmalonyl-CoA to isobutyryl-CoA, it is notable that all residues discussed above are conserved between methylmalonyl-CoA mutase and isobutyryl-CoA mutase of S. cinnamonensis (30).
The model of negative catalysis predicts multiple interactions of a substrate with its enzyme to direct the radical reaction and suppress any unwanted side reactions (41,42). This should in turn require a strict amino acid conservation of the respective protein that would explain why mutases with different substrate specificities strongly cluster together and form distinct subclades ( Fig. 7 and supplemental Fig. S3). This also has consequences for the emergence of mutases with novel substrate specificities. A change in substrate specificity would demand several very specific amino acid substitutions together with conservation of the remaining residues, which in evolutionary terms must be an extremely rare event and likely only occurred once. The following scenario is proposed. During a singular event, ethylmalonyl-CoA and isobutyryl-CoA mutases evolved from an ancestral methylmalonyl-CoA mutase and were distributed by lateral gene transfer. Consistent with this is the fact that methylmalonyl-CoA mutases are widely distributed, and their clustering follows phylogenetic lines. An exception is archaeal methylmalonyl-CoA mutases, which are located in the isobutyryl-CoA mutase cluster. The only reported function of these methylmalonyl-CoA mutases so far is the involvement in a novel pathway of CO 2 fixation (43). One possibility is that an archaeal methylmalonyl-CoA mutase has evolved from an isobutyryl-CoA mutase-like ancestor.
Occurrence and Proposed Function of Ethylmalonyl-CoA Mutase-Genes coding for ethylmalonyl-CoA mutases were found in completely sequenced and fully assembled genomes of a number of bacteria belonging to ␣-proteobacteria, streptomycetes, and two Leptospira species (for a detailed list see Fig.  7B). With the exception of the Frankia and Leptospira species, all bacteria also contain copies of genes encoding the other characteristic enzymes of the ethylmalonyl-CoA pathway: crotonyl-CoA carboxylase/reductase (4), mesaconyl-CoA hydratase (3,5), L-malyl-CoA/␤-methylmalyl-CoA lyase (44), and an acyl-CoA dehydrogenase proposed to encode methylsuccinyl-CoA dehydrogenase. 3 The presence of all those genes suggests a functional ethylmalonyl-CoA pathway in these organisms. In addition, Ccr activity has already been demonstrated in selected representatives, such as R. sphaeroides, M. extorquens, and S. coelicolor, grown on substrates requiring a glyoxylate cycle-independent assimilation pathway (4). In the case of M. extorquens, a well known type II methylotroph lacking the glyoxylate cycle (6,23,45), the ethylmalonyl-CoA pathway is part of C 1 assimilation, whereas in S. coelicolor it functions in the assimilation of butyrate.
In all the bacteria mentioned above, the genes encoding Ecm and Ccr are always clustered on the chromosome, whereas the other genes involved in the pathway are not necessarily grouped. For some Frankia, Salinospora, and Streptomyces species, additional copies of ccr genes are present elsewhere on the genome and are not clustered with ecm but with genes encoding polyketide synthases. The corresponding enzymes likely provide ethylmalonyl-CoA as precursors for the biosynthesis of secondary metabolites, including antibiotics (4,46,47). In these cases, ecm is absent from the gene clusters because methylsuccinyl-CoA does not represent an ␣-alkylmalonyl-CoA ester and, therefore, does not serve as an extender unit for polyketide synthesis (39,48). The presence of ecm in a given genomic context clustering together with ccr therefore strongly indicates an operating acetate assimilation pathway and makes ecm more than ccr a specific marker for the presence of the complete ethylmalonyl-CoA pathway in an organism.
In this context it is noteworthy that genes encoding putative Ecm and Ccr are present in the genomes of the Frankia and Leptospira species listed above, but there they are not grouped in an apparent genetic entity. Furthermore obvious candidates for genes of other characteristic enzymes of the ethylmalonyl-CoA pathway are also missing. Therefore, the possible involvement of ethylmalonyl-CoA mutase in other metabolic pathways in these bacteria as well as the substrates, requiring the ethylmalonyl-CoA pathway in all organisms listed above, remains to be elucidated.