Human B₁₂-dependent enzymes: Methionine synthase and Methylmalonyl-CoA mutase

Abstract

Humans have only two known cobalamin or B₁₂-dependent enzymes: cytoplasmic methionine synthase and mitochondrial methylmalonyl-CoA mutase. A complex intracellular B₁₂ trafficking pathway, comprising a multitude of chaperones, process and deliver cobalamin to the two target enzymes. Methionine synthase catalyzes the transfer of a methyl group from N⁵-methytetrahydrofolate to homocysteine, generating tetrahydrofolate and methionine. Cobalamin serves as an intermediate methyl group carrier and cycles between methylcobalamin and cob(I)alamin. Methylmalonyl-CoA mutase uses the 5′-deoxy-adenosylcobalamin form of the cofactor and catalyzes the 1,2 rearrangement of methylmalonyl-CoA to succinyl-CoA. Two chaperones, CblA (or MMAA) and CblB (or MMAB, also known as adenosyltransferase), serve the mutase and ensure that the fidelity of the cofactor loading and unloading processes is maintained. This chapter focuses on assays for purifying and measuring the activities of methionine synthase and methylmalonyl-CoA mutase.

1. Introduction

Humans have two B₁₂ or cobalamin-dependent enzymes: methionine synthase (MS) and methylmalonyl-CoA mutase (MCM) (Banerjee & Ragsdale, 2003). MS and MCM represent the termination of the cytoplasmic and mitochondrial branches of the B₁₂ trafficking pathway, respectively, that processes and delivers the cofactor to these enzymes (Banerjee, 2006; Banerjee, Gherasim, & Padovani, 2009; Gherasim, Lofgren, & Banerjee, 2013). Redox-linked coordination chemistry plays a key role in the transfer of cobalamin through its intracellular trafficking pathway (Banerjee, Gouda, & Pillay, 2021). MS converts the circulating form of folic acid, N⁵-methytetrahydrofolate (CH₃–H₄ folate), to tetrahydrofolate (H₄ folate), making it available as a one-carbon carrier for essential purine and amino acid biosynthesis reactions (Banerjee & Matthews, 1990). In the process, MS recycles homocysteine to methionine. MS uses the methylcobalamin (MeCbl) form of the cofactor and catalyzes two successive methyl transfer reactions from MeCbl to homocysteine (Eq. 1) and from CH₃–H₄ folate to cob(I)alamin (Eq. 2), generating methionine and H₄folate (Fig. 1).

$$\text{E}•{\text{CH}}_3-\text{cob}(III)\text{ alamin }+\text{homocysteine}\to \text{E}•\text{cob}(I)\text{alamin}+\text{methionine}$$ (1)

$${\text{CH}}_3-{\text{H}}_4\text{folate}+\text{E}•\text{cob}(I)\text{alamin}\to {\text{H}}_4\text{folate}+\text{E}•{\text{CH}}_3-\text{cob}(III)\text{alamin}$$ (2)

Demethylation of MeCbl generates a supemucleophilic cob(I)alamin intermediate (Banerjee, Frasca, Ballou, & Matthews, 1990), which is susceptible to oxidative inactivation approximately once every 2000 turnovers (Drummond, Huang, Blumenthal, & Matthews, 1993). Inactive MS is repaired in a reductive methylation reaction, requiring NADPH-dependent methionine synthase reductase (MSR) as an electron donor and S-adeno-sylmethionine (AdoMet) as a methyl donor (Banerjee, Harder, Ragsdale, & Matthews, 1990; Olteanu, Wolthers, Munro, Scrutton, & Banerjee, 2004). The reactivation cycle results in reformation of MeCbl, enabling reentry of MS to the catalytic cycle (Fig. 1). MSR is a diflavin oxidoreductase that is a dedicated redox partner for MS (Olteanu & Banerjee, 2001). Functional deficiency of either MS (Gulati et al., 1996) or MSR (Leclerc et al., 1998) leads to homocystinuria, an inborn error of metabolism that is inherited as an autosomal recessive disorder (Watkins & Rosenblatt, 2011).

Fig. 1.

Reaction catalyzed by methionine synthase. THF, Hcy and Met denote tetrahydrofolate, homocysteine, and methionine, respectively.

Human MS is a 140 kDa monomeric protein that is predicted to be modular in its organization like its E. coli homolog (Goulding, Postigo, & Matthews, 1997). The N-terminal homocysteine and CH₃–H₄ folate binding domains engage with the central B₁₂ domain during the catalytic cycle, alternately demethylating MeCbl, and remethylating cob(I)alamin, respectively. The C-terminal AdoMet domain engages with the B₁₂ domain during the reactivation cycle.

Human MCM belongs to the class of 5′-deoxyadenosylcobalamin (AdoCbl)-dependent isomerases and catalyzes the 1,2 rearrangement of methylmalonyl-CoA to succinyl-CoA, which can enter the TCA cycle (Fig. 2). The MCM reaction is important in the catabolism of odd-chain fatty acids, branched chain amino acids and cholesterol (Banerjee, 2001, 2003). AdoCbl serves as a radical reservoir and substrate binding accelerates the rate of cobalt-carbon bond cleavage, initiating the radical-based carbon skeleton rearrangement reaction (Chowdhury & Banerjee, 2000) (Fig. 2). The 5′-deoxyadenosine is occasionally lost from the catalytic cycle, leading to cob(II)alamin accumulation in inactive enzyme, which triggers cofactor off-loading (Padovani & Banerjee, 2009a). Both AdoCbl loading onto MCM and cob(II)alamin off-loading from MCM rely on the chaperones CblA and ATR in the mitochondrial B₁₂ trafficking pathway (Fig. 3A and B).

Fig. 2.

Reaction catalyzed by MCM. A histidine ligand donated by MCM serves as the lower axial ligand while the DMB nucleotide tail of the corrin is displaced to the side.

Fig. 3.

AdoCbl loading onto MCM (A) and cob(II)alamin off-loading from MCM (B) require the chaperones ATR and CblA. Redox-linked coordination changes lead to differences in the absorption spectra of cobalamin bound to ATR and MCM, as indicated. The DMB tail is shown attached to the corrin ring.

AdoCbl is synthesized by ATP:cob(I)alamin adenosyltransferase (ATR, also known as MMAB) (Yamanishi, Labunska, & Banerjee, 2005), which also functions as a chaperone, loading AdoCbl onto MCM (Padovani & Banerjee, 2009b; Padovani, Labunska, Palfey, Ballou, & Banerjee, 2008) and off-loading cob(II)alamin from MCM (Padovani & Banerjee, 2009a; Ruetz et al., 2019). AdoCbl bound to MCM is 6-coordinate and has a histidine ligand donated by the protein at the lower axial position. The 5,6 dimethylbenzimidizole (DMB) tail displaced in a side pocket (Froese et al., 2010). In ATR on the other hand, AdoCbl is bound as a 5-coordinate species (Campanello et al., 2018). The vacancy at the lower axial position of AdoCbl together with the DMB tail of the cofactor are important for its translocation from ATR to MCM (Mascarenhas, Ruetz, McDevitt, Koutmos, & Banerjee, 2020). Cofactor movement between MCM and ATR in either direction requires a second chaperone CblA (also known as MMAA). CblA uses both GTP binding and hydrolysis energy to gate the movement of the cofactor (Padovani & Banerjee, 2009a; Ruetz et al., 2019) (Fig. 3). Functional deficiency of CblA (Dobson et al., 2002), ATR (Dobson et al., 2002) or MCM (Ledley, Lumetta, Nguyen, Kolhouse, & Allen, 1988) results in methylmalonic aciduria, an inborn error of metabolism that is inherited as an autosomal recessive disease (Watkins & Rosenblatt, 2011). In this chapter, the purification and assays for MS and MCM are described. Purification and assays for the B₁₂ chaperones ATR and CblA are described in a companion chapter.

1.1. Expression and purification of human MS from insect cells

1.1.1. Expression of MS

Materials and Equipment

• pFastBac Dual LIC cloning vector (Addgene plasmid #30121)

• Bac-to-Bac expression system (ThermoFisher #10359016)

• Spodoptera frugiperda (Sf21) insect cells (Invitrogen)

• High Five^™ Insect Cells (ThermoFisher)

• Insect-Xpress Medium (Lonza)

• Antibiotic-Antimycotic-100 × (Gibco)

• Fisherbrand^™ Shaker Flasks 250 mL (PBV250)

• 2.8 L Nalgene Fernbach Flask

• Hemocytometer

• Biosafety cabinet

• Temperature controlled shaker-incubator that holds 250 mL and 2.8 L flasks

Procedure

1. Human MS cDNA inserted into the pFastBac Dual LIC cloning vector with an N-terminal HisX₆-tag followed by a maltose binding protein (MBP) tag and a TEV cleavage site between MBP and MS, is used for recombinant protein expression

2. Generate recombinant baculovirus for MS expression using the Bac-to-Bac expression system following the manufacturer’s protocol. Generate low titer virus (P1) by transfecting recombinant bacmids into Sf21 cells. Amplify the low titer virus for two more rounds to get a high virus titer (P3) following the manufacturer’s protocol

3. For growth and maintenance of insect cell lines (Sf21 or High Five^™) follow the vendor’s protocols. Briefly, warm Insect-Xpress medium (1 L) to 27 °C in a temperature-controlled water bath and move into biosafety cabinet. Add 10 mL of Antibiotic-Antimycotic to each 1 L bottle and shake well. The cells are maintained at a density ~30–400 × 10⁴ cells/mL in 250 mL flasks at 27 °C. The cell density is estimated everyday by checking cell count using a 10 μL aliquot of the culture in a hemocytometer. If the cell density is >400 × 10⁴ cells/mL, change medium by mixing 18 mL of cell stock with 18 mL fresh medium in a new 250 mL flask

4. For protein expression, prepare a 1 L High Five^™ cell stock with ~300 × 10⁴ cells/mL in Insect-Xpress Medium with 10 mL Antibiotic-Antimycotic −100 ×. From this 1 L cell stock, prepare 6 × 1 L Hi5 cell cultures containing ~70 × 10⁴ cells/mL by diluting with medium (233 mL of 300 × 10⁴ cells/mL with 767 mL medium). Incubate for ~24 h in a 2.8 L sterile flask at 27 °C with shaking at 110 rpm. The cell count should be between 100 and 200 × 10⁴ cells/mL before infecting with 10–50 mL of a high titer P3 virus. Incubate cell cultures for an additional 44 h at 27 °C, 110 rpm

5. Harvest cells by centrifugation at 5500 × g for 30 min in 1 L centrifuge bottles. Transfer the cell pellet into a 50 mL falcon tube and store at −80 °C until use

6. Note: Maintain sterile practices when handling insect cells. All equipment must be autoclaved and sprayed down with 70% ethanol before moving into the bio-safety cabinet. Cover windows on the shaker incubator with aluminum foil to ensure that no light enters during protein expression

1.1.2. Purification of human MS

Materials and Equipment

• Buffer A: 50 mM Tris-HCl pH 8, containing 300 mM NaCl

• Buffer B: 50 mM Tris-HCl pH 8, containing 300 mM NaCl and 10% glycerol

• Lysozyme from chicken egg white (Sigma)

• EDTA-free complete Protease Inhibitor Cocktail (Roche)

• β-mercaptoethanol

• Purified His₆-tagged tobacco etch virus (TEV) protease (1 mg/mL)

• Bio-Rad Protein Assay Kit

• Amylose Resin (New England Biolabs)

• Maltose

• 1 M Dithiothreitol (DTT)

• Superdex 200 (S200, 120 mL, GE Healthcare)

• Amicon Ultra-15 50 kDa centrifugal filters (Millipore-Sigma)

Procedure

1. Thaw and suspend the Hi5 cell pellet infected with recombinant baculovirus in 400 mL Buffer A containing 1 tablet of EDTA-free complete Protease Inhibitor Cocktail, 20 mg of lysozyme and 3 mM β-mercaptoethanol. Divide the cell suspension into 50 mL falcon tubes and lyse cells by 4 cycles of freezing in liquid N₂ and thawing in a water bath at room temperature

2. Centrifuge the cell lysates at 38,000 × g at 4 °C for 2 h

3. Pool the supernatant in a beaker and load onto an amylose resin column (2.5 × 5 cm) equilibrated with Buffer B at 4 °C and then wash with 200 mL Buffer B

4. Elute His-MBP-tagged MS with Buffer B containing 10 mM maltose and collect 10 mL fractions. Measure the 595 nm absorbance using 5 μL of each fraction and 200 μL of Bio-Rad Protein Assay Reagent in a 96-well plate. Use 0.3 absorbance units as a cut-off to pool fractions containing His-MBP-tagged MS. The purity of His-MBP-tagged MS is shown in Fig. 4A

5. Concentrate His-MBP-tagged MS to 20 mL using Amicon Ultra-15 (MWCO = 50 kDa) filters. Transfer the concentrated protein to an appropriate length of SnakeSkin Dialysis Tubing (MWCO = 10 kDa) and add 1 mL of TEV-protease. Dialyze (overnight at 4 °C) with stirring in a 500 mL beaker containing 400 mL Buffer B supplemented with 0.4 mL DTT

6. Load the supernatant onto an amylose column (2.5 × 5 cm) and wash with Buffer B. Pool the flow-through and wash and concentrate tag-less MS to 5 mL using Amicon Ultra-15 (MWCO = 50 kDa) filters

7. Load MS onto a Superdex 200 column (S200, 100 mL, GE Healthcare) equilibrated with Buffer B. Monitor MS-containing fractions by their absorbance at 280 nm, pool and concentrate to 0.5 mL using an Amicon Ultra-15 50 kDa centrifugal filter

8. Estimate the concentration of MS concentration using ε₂₈₀ = 116,550 M⁻¹ cm⁻¹. Separate 25 μL aliquots in 750 μL microcentrifuge tubes, flash-freeze in liquid nitrogen and store at −80 °C till further use. A typical yield is ~3–5 mg MS from 6 L culture

Fig. 4.

SDS-PAGE gel analysis of purified methylmalonyl-CoA mutase(A) and maltose binding protein-tagged methionine synthase (B).

1.2. Assays for methionine synthase activity

Several methods have been developed for measuring the activity of MS (Chen, Chakraborty, & Banerjee, 1995; Drummond, Jarrett, Gonzalez, Huang, & Matthews, 1995; Frasca, Banerjee, Dunham, Sands, & Matthews, 1988; Yamada, Strahler, Andrews, & Matthews, 2005). Here, we describe two assays. In the first, transfer of the methyl group from 5-¹⁴CH₃-H₄ folate to the product methionine, is monitored. In the second, the product H₄ folate, is derivatized with a formylating agent under acidic conditions to generate methenyl-H₄ folate (CH⁺ = H₄folate), which is detected at 350 nm (Eq. 3). Unused 5-CH₃-H₄ folate does not contribute to the absorbance at this wavelength.

$${\text{H}}_4\text{folate}+\text{HCOOH}\to {\text{CH}}^{+}={\text{H}}_4\text{folate}+2{\text{ H}}_2\text{O}$$ (3)

1.2.1. Radioactive MS assay

Materials

• 5 mM ¹⁴CH₃-H₄ folate (1200 dpm/nmol)

• 1 mM AdoMet

• 25 mM homocysteine

• 1 M dithiotreitol (DTT)

• 0.5 mM aquocobalamin (OH₂Cbl)

• 0.2 M potassium phosphate buffer, pH 7.2

• AG 1-X8 anion exchange resin (Bio Rad)

Procedure

Prepare a 250 μL reaction by mixing 10 μL homocysteine, 25 μL AdoMet, 6.25 μL DTT, 25 μL OH₂Cbl and MS in 0.2 M potassium phosphate buffer pH 7.2. Incubate at 37 °C for 5 min in a temperature-controlled water bath. Start the reaction by adding 12.5 μL ¹⁴CH₃-H₄ folate and incubate at 37 °C for 10 min. Quench at 90 °C for 3 min to terminate the reaction followed by cooling on ice for 2 min. Load 200 μL onto a 0.5 × 6 cm column of Bio-Rad AG 1-X8 (chloride form). Wash the column with 1.5 mL water and measure ¹⁴C radioactivity associated with methionine in the combined flow-through and wash using a scintillation counter. One unit of MS is defined as the amount of enzyme required to catalyze the formation of 1 μmol of methionine/min at 37 °C.

1.2.2. Non-radioactive MS assay

Materials

• 1 M potassium phosphate buffer, pH 7.2

• 500 mM DTT

• 3.8 mM AdoMet

• 100 mM L-homocysteine

• 500 μM OH₂Cbl

• 250 μM (6R,S)-CH₃-H₄ folate

• 5 N HCl in aqueous 60% formic acid

Procedure

Prepare reagents as described by Drummond et al. (Drummond et al., 1995). Combine distilled water (544 μL, less the volume of enzyme to be added) with the following reagents in the given order: 80 μL potassium phosphate buffer, pH 7.2, 40 μL DTT, 4 μL AdoMet, 4 μL L-homocysteine, and MS. Prepare a blank lacking MS. Next, add 80 μL of OH₂Cbl and incubate at 37 °C. DTT and OH₂Cbl catalyze the reduction of O₂ to H₂O₂, creating a semi-anaerobic environment. Timing is critical from this point onwards, as the time required for activation of MS must be balanced by its slow inactivation as H₂O₂ builds up. After exactly 5 min, add 48 μL of (6R,S)-CH₃–H₄ folate to initiate turnover and incubate at 37 °C for 10 min. The concentration of H₄ folate must fall within the linear range of the assay, optimally between 0.1 and 1 absorbance units at 350 nm. Add 200 μL of the acidic derivatization solution (5 N HCl in aqueous 60% formic acid) to quench the reaction and heat immediately at 80 °C for 10 min to convert H₄ folate to CH⁺ = H₄ folate. Cool the reaction mixture to room temperature and record the absorbance in a spectrophotometer, with a temperature-controlled cuvette holder maintained at 4 °C. The concentration of CH⁺ = H₄ folate is determined using Δε₃₅₀ = 26,500 M⁻¹ cm⁻¹.

2. Expression and purification of MCM

2.1. Expression of MCM

Materials

• E. coli expression strain BL21(DE3)

• Terrific broth (TB) and Luria Bertani (LB) media

• LB agar plates containing 50 μg/mL of kanamycin

• Dimethyl sulfoxide (DMSO)

• Kanamycin, 50 mg/mL

• 1 M IPTG

• Erlenmeyer flasks (6 × 2.8 L), temperature-controlled shaker

Procedure

1. The human MCM gene was codon optimized for expression in E. coli and cloned into a pET-28b(+) vector with a TEV cleavable C-terminal His₆ tag between restriction sites Nco1 and Xho1 (Campanello et al., 2018)

2. Transform BL21(DE3) cells with the pET28b(+)-MCM vector and plate on LB agar containing 50 μg/mL kanamycin. Incubate at 37 °C overnight

3. Inoculate 10 mL sterilized LB medium containing 50 μg/mL kanamycin with a single colony and incubate at 37 °C overnight with shaking at 220 rpm

4. Inoculate culture flasks (6 × 2.8 L) containing 1 L sterilized TB medium and 50 μg/mL of kanamycin with 1 mL of the overnight culture. Incubate at 37 °C with shaking at 220 rpm until OD₆₀₀ = 1.5–1.8 (in ~6–8 h)

5. Add 30 mL of DMSO to 1 L culture (3% final concentration) and cool the culture to 16 °C by shaking for 40 min

6. Induce expression of MCM by adding 0.25 mL IPTG (250 μM final concentration) to each flask and incubate at 16 °C overnight (22–24 h) with shaking at 220 rpm

7. Pellet cells by centrifugation at 5500 × g for 20 min in 1 L centrifuge bottles. Transfer cell pellet into 50 mL falcon tubes and store at −80 °C until use. A typical yield is 80–100 g of wet cell pellet from a 6 L culture

2.2. Purification of MCM

Materials

• Misonix Sonicator (Model XL, 1.2 cm diameter probe)

• Temperature-controlled centrifuge

• Ni-NTA resin (Qiagen)

• Amicon 50 kDa centrifugal tube (Millipore)

• Ion exchange column: Source Q (Omnifit)

• FPLC system (GE Healthcare)

• His-tagged TEV protease (Sigma)

• Complete EDTA-free protease inhibitor cocktail tablet (Roche)

• 100 mM Phenylmethylsulfonyl fluoride in DMSO

• Lysis buffer: 50 mM Tris pH 8.0, 500 mM NaCl, 20 mM imidazole and 5% glycerol

• TEV buffer: 50 mM Tris pH 8.0, 300 mM NaCl, 0.5 mM EDTA, 1 mM DTT and 5% glycerol

• Buffer A: 50 mM HEPES pH 7.3, 25 mM NaCl and 5% glycerol

• Buffer B: 50 mM HEPES pH 7.3, 500 mM NaCl and 5% glycerol

• Storage Buffer: 50 mM HEPES pH 7.5, 150 mM KC1, 2 mM TCEP, 2 mM MgCl₂, 5% glycerol

Procedure

1. Thaw the cell pellet and resuspend in Lysis Buffer (6–8 mL per g of cell pellet) in a 500 mL beaker. Add 0.1 mL PMSF per 100 mL of cell suspension (100 μM final concentration) and one protease inhibitor cocktail tablet and stir at 4 °C for 30 min

2. Lyse cells by sonication using a 1.2 cm diameter probe at 30% power with cycles (15 s on, 1 min off) for a total of 20 min on ice

3. Transfer the cell lysate to 50 mL tubes and centrifuge (38,500 × g for 45 min, 4 °C)

4. Load the supernatant onto a Ni-NTA column and wash with 150 mL Lysis Buffer. Elute His₆-MCM using a gradient mixer containing 150 mL Lysis Buffer and 150 mL Lysis Buffer with 200 mM imidazole

5. Check protein concentration by mixing 10 μL of each fraction with 990 μL of the Bradford reagent in a plastic cuvette and measure the absorbance at 595 nm. Pool and concentrate fractions using an Amicon centrifugal device (50 kDa cut-off)

6. Add His-tagged TEV protease (0.02 mg/mg) to His₆-MCM in a dialysis bag (10 kDa cut-off) and dialyze overnight in 1 L TEV Buffer at 4 °C to cleave the N-terminal His₆-tag on MCM

7. The following day, add additional TEV protease (0.01 mg/mg) to the protein in the dialysis bag and dialyze against TEV buffer for an additional 3–4 h

8. Load the TEV treated MCM solution onto a Ni-NTA column (equilibrated with TEV Buffer) to remove His-tagged TEV. Collect the flow through and wash the column with Lysis Buffer (100–150 mL) until no protein is present in the flow through as detected by the Bradford assay

9. Combine flow through and wash fractions containing MCM and exchange with Buffer A using an Amicon centrifugal device (MWCO = 50 kDa). Alternatively, concentrate MCM to 20–30 mL and dialyze against 1 L Buffer A overnight at 4 °C

10. Equilibrate the SourceQ column (Omnifit) with Buffer A on an FPLC system. Load the protein onto the column and wash with 50 mL Buffer A. MCM is then eluted with the following gradient: 0–70% Buffer B in 35 min, followed by 100% Buffer B in 1 min and maintained at 100% Buffer B for 5 min. Monitor absorbance at 280 nm and collect 5 mL fractions. Concentrate and dialyze MCM-containing fractions against 1 L Storage Buffer overnight at 4 °C

11. Concentrate MCM to 2–3 mL and determine the MCM (dimer) concentration using ε_280nm = 133,620 M⁻¹ cm⁻¹. Aliquot, flash freeze in liquid N₂ and store at −80 °C. A typical yield is ~90 mg MCM from a 6 L culture. The purity of MCM is shown in Fig. 4B

    Note: Alternatively, a Mono Q column (GE Healthcare) can be used with the same gradient.

2.3. Determination of MCM activity

MCM activity is determined in a coupled enzyme assay with thiokinase (also known as succinyl-CoA synthase). Thiokinase catalyzes the conversion of succinyl-CoA to CoA and succinic acid in the presence of GDP and phosphate (Fig. 5). The free thiol in CoA is detected using Ellman’s reagent, 5,5-dithio-bis-(2-nitrobenzoic acid or DTNB) and the concentration of succinyl-CoA formed in the MCM reaction is estimated using ε_412nm = 14,150 M⁻¹ cm⁻¹ (Ellman, 1959).

Fig. 5.

MCM activity as detected in the coupled thiokinase assay.

Materials

• Buffer A: 100 mM potassium phosphate, 3 mM MgCl₂, pH 7.5

• 1 M potassium phosphate pH 7.5

• 100 mM magnesium chloride (MgCl₂)

• 100 mM guanosine diphosphate (GDP)

• 5 mg/mL succinyl-CoA thiokinase (Sigma)

• 10 mM DTNB in ethanol

• 50 mM methylmalonyl-CoA in water

• 50 μM MCM dimer

• 500 μM AdoCbl

Procedure

The catalytic activity of MCM is monitored using a UV–visible spectrophotometer with a temperature-controlled cuvette holder. In a 1.5 mL Eppendorf tube, add 38 μL Buffer A, 2 μL AdoCbl and 10 μL MCM to prepare a 10 μM stock of holo-MCM. Incubate at 30 °C using a temperature-controlled Eppendorf Thermostat. After 15 min, transfer the sample to ice. Dilute the sample 1:100 by adding 2 μL of MCM to 198 μL Buffer A to obtain a 100 nM stock of holo-MCM.

In a quartz cuvette, add 178.6 μL Buffer A, 6 μL GDP, 1.4 μL DTNB, 2 μL AdoCbl and 4 μL of a 1:1 (v/v) dilution of the methyl-malonyl-CoA stock with 1 M potassium phosphate pH 7.5 (see note below). Place the cuvette in a spectrophotometer for 10 min at 30 °C. Add 4 μL thiokinase and monitor the change in absorbance at 412 nm for 5 min to determine background rate. Initiate the reaction by adding 4 μL of 100 nM holo-MCM (2 nM final concentration) and record the increase in absorbance at 412 nm absorbance. The initial rate is used to determine MCM activity using ε_412nm = 14,150 M⁻¹ cm⁻¹ after subtracting the background thiokinase rate. The specific activity of MCM is typically 67 ± 2 μM succinyl-CoA formed min⁻¹ mg⁻¹ at 30 °C.

Note: The methylmalonyl-CoA stock solution is acidic and the 1:1 mixture with 1 M potassium phosphate pH 7.5, is made immediately before use in the assay.

2.4. Assay for AdoCbl loading onto MCM

ATR is tasked with both the synthesis and delivery of AdoCbl to the mitochondrial target, MCM. The transfer can be readily monitored by the large absorbance change that accompanies the movement of 5-coordinate AdoCbl from ATR to 6-coordinate AdoCbl bound to MCM (Fig. 3). AdoCbl transfer is gated by the GTPase CblA. The non-hydrolysable GTP analog β,γ-methyleneguanosine 5′-triphosphate (GMPPCP) can be used as a GTP mimic (Ruetz et al., 2019) (Fig. 6A).

Fig. 6.

Spectral analysis of cofactor translocation. (A) Transfer of AdoCbl from ATR (5-coordinate base-off) to MCM (6-coordinate base-off, His-on). (B) Transfer of cob(II) alamin from MCM (5-coordinate) to ATR (4-coordinate) in the presence of ATP. Buffer A was used in these experiments.

Materials

• Buffer A: 50 mM HEPES pH 7.4, 150 mM KC1, 2 mM TCEP, 2 mM MgCl₂, 5% glycerol

• 250 μM MCM

• 500 μM ATR

• 1 mM AdoCbl

• 750 μM CblA

• 100 mM GTP

• 100 mM GMPPCP

Note: Commercial GTP is sometimes contaminated with PPPi, which leads to formation of the 440 nm species under anaerobic conditions (corresponding to a weakened cobalt-carbon bond in AdoCbl) or to the homolytic cleavage of this bond under aerobic conditions (Campanello et al., 2018).

Equipment

UV/visible spectrophotometer with a temperature-controlled cuvette holder

Ultra-micro quartz glass cuvette

Note: GMPPCP or β,γ-imidoguanosine 5′-triphosphate (GMPPNP).

Procedure

In a quartz cuvette, add 165 μL, Buffer A, 3 μL. AdoCbl and 6 μL. ATR and record the spectrum between 300 and 700 nm. Estimate the concentration of ATR bound AdoCbl using ε_455nm = 8000 M⁻¹ cm⁻¹ (Fig. 6A). Place the cuvette in a spectrophotometer for 10 min at 25 °C. Meanwhile, mix 12 μL, MCM, 12 μL, CblA and 2 μL, GTP or 2 μL. GMPPCP in a 0.5 ml Eppendorf tube. Initiate the transfer by adding 26 μL of the MCM-CblA-GTP or MCM-CblA-GMPPCP mixture to the cuvette and record the spectrum every minute for 40 min. In the presence of GTP, an increase at 525 nm corresponding to AdoCbl transfer to MCM is observed, whereas GMPPCP inhibits transfer, resulting in a slight increase at 525 nm. Plot the change in absorbance at 525 nm and fit the data to a single exponential to determine the rate of AdoCbl transfer from ATR to MCM. The concentration of AdoCbl transferred from ATR to MCM is determined using Δε _525nm = 7500 M⁻¹ cm⁻¹.

2.5. Assay for cob(II)alamin off-loading from MCM to ATR

In the catalytic cycle of MCM, the 5′deoxyadeonsyl radical is occasionally lost, resulting in inactive cob(II)alamin on MCM (Padovani & Banerjee, 2009a). Cob(II)alamin is transferred back to ATR for repair with the help of the CblA chaperone in the presence of GTP (Fig. 3). The UV/visible spectrum of cob(II)alamin bound to MCM is indistinguishable from that of 5-coordinate cob(II)alamin in solution (λ_max = 474 nm), whereas ATR bound cob(II)aLamin is 4-coordinate in the presence of ATP (λmax = 464 nm) (Fig. 6B).

Materials

• Buffer A: 50 mM HEPES pH 7.4, 150 mM KC1, 2 mM TCEP, 2 mM MgCl₂, 5% glycerol

• 1 mM cob(II)alamin

• 200 μM MCM

• 600 μM CblA

• 500 μM ATR

• 100 mM GTP

• 250 mM ATP

Note: All solutions are made anaerobic by purging with N₂ for 15 min prior before transferring to an anaerobic chamber.

Method

Prepare 2 mL of a 1 mM AdoCbl stock solution in a glass cuvette sealed with a rubber stopper and parafilm and purge with N₂ gas for 15 min. Then, place the AdoCbl sample on ice 10 cm away from a halogen lamp (38 W, G.E.) After 2–3 h, move the sample into an anaerobic chamber. Complete photolysis of AdoCbl is confirmed by the absorption spectrum of cob(II)alamin (λ_max = 473nm, ε_473nm = 9.2 mM⁻¹ cm⁻¹).

Note: The pH of the ATP stock, which is acidic, is adjusted to 7.5 with 1 M HEPES pH 7.5.

Equipment

• Anaerobic chamber (<0.4 O₂ ppm)

• UV/visible spectrophotometer with a temperature-controlled cuvette holder

• Ultra-micro UV/visible quartz glass cuvette

Procedure

Cob(II)alamin off-loading from MCM (Fig. 6B) is monitored using a UV–visible spectrophotometer in an anaerobic chamber with a temperature-controlled cuvette holder set to 25 °C. In a quartz cuvette, add 166 μL Buffer A and 3 μL. of cob(II)alamin. Add 9 μL MCM and incubate the cuvette in the spectrophotometer for 15 min. In a 0.5 mL Eppendorf tube, prepare the “repair mixture” by adding 4 μL ATP, 6 μL of ATR, 10 μL CblA and 2 μL GTP. Initiate the cob(II)alamin transfer reaction by adding the repair mixture to the cuvette. Record the spectrum between 300 and 750 nm every 15 s for 20 min. Transfer of cob(II)alamin from MCM to ATR results in an increase in absorbance at 464 nm (ε_464nm = 14,100 M⁻¹ cm⁻¹) (Campanello et al., 2018), which corresponds to 4-coordinate cob(II)alamin bound to ATR. Fit the change in absorbance at 464 nm to a single exponential equation to determine the rate of cob(II)alamin transfer from MCM to ATR. Estimate the concentration cob(II)alamin transferred using Δε_464nm = 5900 M⁻¹ cm⁻¹.

Note: If a spectrophotometer inside an anaerobic chamber is not available, the anaerobic reaction mixture is prepared in a cuvette sealed with a rubber septum and the anaerobic repair mixture prepared separately, is added with a gas-tight Hamilton syringe.