human 5-methyltetrahydrofolate-homocysteine methyltransferase, the homocysteine and folate binding domains

Target ID:

MTRA

Aliases:

FLJ33168FLJ43216FLJ45386MSMTR

Deposition Date:

Tuesday 29th October 2013

Families:

Miscellaneous

Structure type:

apo

Download Coordinates:

4CCZ

Authors:

M.Vollmar, W.Kiyani, T.Krojer, S.Goubin, N.Burgess-Brown, F.von Delft, U.Oppermann, A.Edwards, C.Arrowsmith, C.Bountra, W.W.Yue

Site:

Oxford

4CCZ

tabs

Structure Details

Methionine synthase (EC 2.1.1.13; also known as 5-methyltetrahydrofolate-homocysteine methyltransferase, MTR) is a multi-domain, cobalamin-dependent enzyme that catalyzes the conversion of homocysteine (Hcy) to methionine. MTR plays a key role in folate metabolism, and is integral to the methionine cycle that recycles Hcy to generate the universal methyl donor, S-adenosyl-methionine (AdoMet). Inherited mutations in the human mtr gene (chromosome location 1p43) cause the cblG type of rare cobalamin disorders [1], characterized by homocystinuria, hyperhomocysteinemia, and hypomethioninemia.

The reaction catalysed by MTR requires cobalamin (in its nucleophilic state, cob(I)alamin) as well as 5-methyl-tetrahydrofolate (MTHF) as enzyme cofactors. This reaction proceeds by a transfer of the methyl group from MTHF to cob(I)alamin forming the methyl-cob(III)alamin intermediate, which subsequently transfers the methyl group to Hcy yielding the product methionine and regenerating cob(I)alamin [2]. For every 200-1000 MTR turnovers [3], the unstable cob(I)alamin is oxidized to an unreactive cob(II)alamin state, which is reactivated to methyl-cob(III)alamin for catalysis, by a reaction that involves an NADPH-derived electron, the methyl-donor AdoMet, and the reactivating enzyme methionine synthase reductase, MSR.

The complex chemistry underlying the MTR catalysis and reactivation cycles is reflected in the highly modular organization of the protein, which contains four functional domains that can bind their respective ligands Hcy, THF, cobalamin and AdoMet during the catalytic cycle. Here we determined the crystal structure of an N-terminal fragment of MTR harbouring the Hcy and THF binding modules (aa 16 -657) to 2.7 Å.

Materials and Methods

MTR

PDB:4CCZ

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:synthetic gene
SGC Clone Accession:
Tag:N-terminal, TEV protease cleavable hexahistidine tag
Host:

Construct

Prelude:
Sequence:MHHHHHHSSGVDLGTENLYFQ*SMKTLRDEINAILQKRIMVLDGGMGTMIQREKLNEEHFRGQEFKDHARPLKGNNDILSITQPDVIYQIHKEYLLAGADIIETNTFSSTSIAQADYGLEHLAYRMNMCSAGVARKAAEEVTLQTGIKRFVAGALGPTNKTLSVSPSVERPDYRNITFDELVEAYQEQAKGLLDGGVDILLIETIFDTANAKAALFALQNLFEEKYAPRPIFISGTIVDKSGRTLSGQTGEGFVISVSHGEPLCIGLNCALGAAEMRPFIEIIGKCTTAYVLCYPNAGLPNTFGDYDETPSMMAKHLKDFAMDGLVNIVGGCCGSTPDHIREIAEAVKNCKPRVPPATAFEGHMLLSGLEPFRIGPYTNFVNIGERCNVAGSRKFAKLIMAGNYEEALCVAKVQVEMGAQVLDVNMDDGMLDGPSAMTRFCNLIASEPDIAKVPLCIDSSNFAVIEAGLKCCQGKCIVNSISLKEGEDDFLEKARKIKKYGAAMVVMAFDEEGQATETDTKIRVCTRAYHLLVKKLGFNPNDIIFDPNILTIGTGMEEHNLYAINFIHATKVIKETLPGARISGGLSNLSFSFRGMEAIREAMHGVFLYHAIKSGMDMGIVNAGNLPVYDDIHKELLQLCEDLIWNKDPEATEKLLRYAQTQGTGGMHHHHHHSSGVDLGTENLYFQ*SM is the His6 tag folowed by TEV protease recognition site *.
Vector:pNIC28-Bsa4

Growth

Medium:
Antibiotics:
Procedure:

Purification

Buffers
Procedure
Cell Lysis Cell pellets were dissolved in approximately 50ml lysis buffer and broken by passing through a high pressure homogenizer at 15,000 psi for 4 cycles. The cell debris was pelleted at 35,000 x g and the supernatant used for further purification. Lysis Buffer: 50 mM Hepes pH 7.5, 500 mM NaCl, 5% Glycerol, 20 mM Imidazole pH 7.5, 0.5 mM TCEP, 1 tablet per 50 ml protease inhibitor cocktail EDTA-free (Roche) Step 1 Ni-NTA affinityThe clarified supernatant was collected and incubated with 1.5 mL of Ni-NTA in cold room under rotation for 1h30min. The resin was then washed in a glass column with Binding Buffer (2x15 ml) and Wash Buffer (2x15 ml). The protein was eluted with 5x1 ml of Elution Buffer. All steps were done in cold room. Binding Buffer: 50 mM Hepes pH 7.5, 500 mM NaCl, 5% Glycerol, 20 mM Imidazole pH 7.5, 0.5 mM TCEP Wash Buffer: 50 mM Hepes pH 7.5, 500 mM NaCl, 5% Glycerol, 40 mM Imidazole pH 7.5, 0.5 mM TCEP Elution Buffer: 50 mM Hepes pH 7.5, 500 mM NaCl, 5% Glycerol, 250 mM Imidazole pH 7.5, 0.5 mM TCEP Step 2 Superdex 200 16/60 Gel FiltrationEluted fractions were concentrated to 5ml and loaded onto a Superdex 200 16/60 column (pre-equilibrated in GF Buffer) at 1.0 ml/min. 1.75 ml fractions were collected. GF Buffer: 10 mM Hepes pH 7.5, 500 mM NaCl, 0.5 mM TCEP, 5% Glycerol. Step 3 His-tag removalThe N-terminal His6-tag was cleaved by incubating overnight with TEV (20°C). Cleaved protein was purified by batch binding on 1ml pre-equilibriated Ni-NTA beads. The column was then washed with 2x1ml GF buffer, 2x1ml Wash buffer. Step 4 HiTrap Q HP Ion Exchange Cleaved protein was concentrated to 5 mL, and diluted with Zero Salt Buffer to a final NaCl concentration of 50 mM. The diluted sample was loaded onto the Hitrap Q HP anionic exchange column that was pre equilibrated with Low Salt Buffer. Elution was performed with a linear NaCl gradient from 0% to 25% of High Salt Buffer in 150 ml. 1.75 mL fractions were collected. Zero Salt Buffer: 50 mM HEPES pH 7.5, 5% Glycerol. Low Salt Buffer: 50 mM HEPES pH 7.5, 5% Glycerol, 50 mM NaCl. High Salt Buffer: 50 mM HEPES pH 7.5, 5% Glycerol, 2 M NaCl.

Extraction

Buffers
Procedure
Expression strain BL21(DE3)-R3-pRARE2Transformation The construct DNA was transformed into competent cells of the expression strain by a standard heat shock procedure. Glycerol stock preparation One colony from the transformation was used to inoculate 1 ml of TB media containing 50 μg/ml kanamycin and 50 μg/ml chloramphenicol, which was placed in a 37°C shaker overnight. The next day glycerol stocks were prepared from this overnight culture. Expression A glycerol stock was used to inoculate 100 ml of TB media containing 50 μg/ml kanamycin and 50 μg/ml chloramphenicol, which was placed in a 37°C shaker overnight. The next day this starter culture was used to inoculate 9 L of TB media (7.5 ml starter culture used per 1L) containing 50 μg/ml kanamycin. When the OD600 reached approximately 0.8 the temperature was reduced to 18°C and after a further 1 hour the cells were induced by the addition of 0.1 mM IPTG. The expression was continued overnight. Cell harvest Cells were harvested by centrifugation at 6000 x g after which the supernatant was poured out and the cell pellet either placed in a -20°C freezer or used directly for purification.
Concentration:The purified protein was concentrated to 14.7 mg/ml using Millipore 30k mwco concentrators.
Ligand
MassSpec:Measured mass: 70447.5 Da Expected mass: 70446.3 Da
Crystallization:Crystals were grown by the sitting drop vapour diffusion method at 20°C. A sitting drop consisting of 75 nl protein and 75 nl well solution was equilibrated against well solution containing 50 mM HEPES pH 7.5, 5% Glycerol. Crystals were mounted in the presence of 25 % (v/v) ethylene glycol and flash-cooled in liquid nitrogen.
NMR Spectroscopy:
Data Collection:Resolution: 2.7 Å X-ray source: Diamond Light Source beamline IO2
Data Processing:

References

Leclerc D, Campeau E, Goyette P, Adjalla CE, Christensen B, Ross M, Eydoux P, Rosenblatt DS, Rozen R and Gravel RA (1996) Human methionine synthase, cDNA cloning and identification of mutations in patients of the cblG complementation group of folate/cobalamin disorders. Hum Mol Genet 5, 1867 - 1874.
Banerjee RV, Frasca V, Ballou DP & Matthews RG (1990) Participation of cob(I)alamin in the reaction catalyzed by methionine synthase from Escherichia coli: a steady-state and rapid reaction kinetic analysis. Biochemistry 29, 11101-11109.
Fujii K, Galivan JH & Huennekens FM (1977) Activa-tion of methionine synthase: further characterization of flavoprotein system. Arch Biochem Biophys 178, 662-670.