Photoaffinity labeling of methylenetetrahydrofolate reductase with 8-azido-S-adenosylmethionine.

Sumner, James S.; Jencks, David A.; Khani, Shahrokh C.; Matthews, Rowena G.

doi:10.1016/s0021-9258(19)57456-7

Cited by 55 publications

(17 citation statements)

References 8 publications

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“…For methionine synthase, the specific residue(s) involved have not been characterized, although labeling is presumed to involve a methyl transfer from [methyl^H] AdoMet to the enzyme. When [35S] AdoMet of the same specific radioactivity was synthesized by the method of Sumner et al (1986) and the cross-linking repeated, labeling by [35S] AdoMet was barely detectable in comparison with labeling by tritiated AdoMet (Christropher Harris, unpublished observation). This is consistent with the observation that methyl group transfer is the mode of labeling for the £coRII and CheR methyltransferases cited above, although the mechanism of this process is not well understood.…”

Section: Resultsmentioning

confidence: 99%

Assignment of enzymic function to specific protein regions of cobalamin-dependent methionine synthase from Escherichia coli

Drummond

Huang

Blumenthal³

et al. 1993

Biochemistry

119

125

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Cobalamin-dependent methionine synthase catalyzes methyl group transfer from methyltetrahydrofolate to homocysteine to form tetrahydrofolate and methionine, and the cobalamin prosthetic group serves as an intermediate methyl carrier. Enzyme possessing cobalamin in the cobalt(II) oxidation state is inactive, and this form is activated by one-electron reduction coupled to methylation by S-adenosylmethionine (AdoMet). The enzyme from Escherichia coli has been divided into separable fragments by limited proteolysis with trypsin, and the contribution of each of these fragments to substrate binding and catalysis has been evaluated. The 37.7-kDa carboxyl-terminal domain binds AdoMet, and this was demonstrated through covalent modification with radiolabeled AdoMet during ultraviolet irradiation. Following reductive activation with AdoMet, the enzyme was digested with trypsin and a 98.4-kDa amino-terminal fragment was isolated. It retained at least 70% of the activity of the intact enzyme and must therefore possess determinants sufficient for the binding of methyltetrahydrofolate and homocysteine, as well as residues required for catalysis. However, when the cobalamin was oxidized to the cob(II) alamin state, the 98.4-kDa fragment could not be reductively remethylated with AdoMet. A purified, 28-kDa domain within the 98.4-kDa fragment retained bound cobalamin and therefore must play a central role in catalysis, but the isolated 28-kDa domain retained no catalytic activity. Because AdoMet binds to a different domain of the protein than methyltetrahydrofolate and homocysteine, the enzyme probably uses conformational flexibility to allow the cobalamin access to the required methyl donor or acceptor at the appropriate time in catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Resultsmentioning

confidence: 99%

Assignment of enzymic function to specific protein regions of cobalamin-dependent methionine synthase from Escherichia coli

Drummond

Huang

Blumenthal³

et al. 1993

Biochemistry

119

125

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“…The enzymes from pig liver and from Homo sapiens are homodimers of 70−77-kDa chains, each consisting of a catalytic domain and regulatory regions that mediate allosteric responses to S -adenosylmethionine ( S -AdoMet) ( , ). These mammalian enzymes are specific for NADPH ().…”

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confidence: 99%

Structures of NADH and CH₃-H₄Folate Complexes of Escherichia coli Methylenetetrahydrofolate Reductase Reveal a Spartan Strategy for a Ping-Pong Reaction^,

2005

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Methylenetetrahydrofolate reductases (MTHFRs; EC 1.7.99.5) catalyze the NAD(P)H-dependent reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate) using flavin adenine dinucleotide (FAD) as a cofactor. The initial X-ray structure of Escherichia coli MTHFR revealed that this 33-kDa polypeptide is a (betaalpha)(8) barrel that aggregates to form an unusual tetramer with only 2-fold symmetry. Structures of reduced enzyme complexed with NADH and of oxidized Glu28Gln enzyme complexed with CH(3)-H(4)folate have now been determined at resolutions of 1.95 and 1.85 A, respectively. The NADH complex reveals a rare mode of dinucleotide binding; NADH adopts a hairpin conformation and is sandwiched between a conserved phenylalanine, Phe223, and the isoalloxazine ring of FAD. The nicotinamide of the bound pyridine nucleotide is stacked against the si face of the flavin ring with C4 adjoining the N5 of FAD, implying that this structure models a complex that is competent for hydride transfer. In the complex with CH(3)-H(4)folate, the pterin ring is also stacked against FAD in an orientation that is favorable for hydride transfer. Thus, the binding sites for the two substrates overlap, as expected for many enzymes that catalyze ping-pong reactions, and several invariant residues interact with both folate and pyridine nucleotide substrates. Comparisons of liganded and substrate-free structures reveal multiple conformations for the loops beta2-alpha2 (L2), beta3-alpha3 (L3), and beta4-alpha4 (L4) and suggest that motions of these loops facilitate the ping-pong reaction. In particular, the L4 loop adopts a "closed" conformation that allows Asp120 to hydrogen bond to the pterin ring in the folate complex but must move to an "open" conformation to allow NADH to bind.

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“…HCy was formed from its thiolactone as described by Hatch et al (1961). [arfe«ojy/-U-14C]AdoMet was prepared enzymatically from [U-14C]ATP and methionine as described by Sumner et al (1986). AdoMet was purified on a Bio-Rex 70 column (Sumner et al, 1986) prior to use in the formation of methylated enzyme.…”

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confidence: 99%

“…[arfe«ojy/-U-14C]AdoMet was prepared enzymatically from [U-14C]ATP and methionine as described by Sumner et al (1986). AdoMet was purified on a Bio-Rex 70 column (Sumner et al, 1986) prior to use in the formation of methylated enzyme. The R and F proteins were purified from E. coli B by methods adapted from those described by Fujii and Huennekens (1974), as outlined by Frasca (1986).…”

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confidence: 99%

Cobalamin-dependent methionine synthase from Escherichia coli B: electron paramagnetic resonance spectra of the inactive form and the active methylated form of the enzyme

et al. 1988

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Cobalamin-dependent methionine synthase (5-methyltetrahydrofolate-homocysteine methyltransferase, EC 2.1.1.13) has been isolated from Escherichia coli B in homogeneous form. The enzyme is isolated in an inactive form with the visible absorbance properties of cob(II)alamin. The inactive enzyme exhibits an electron paramagnetic resonance (EPR) spectrum at 38 K that is characteristic of cob(II)alamin at acid pH, where the protonated dimethylbenzimidazole substituent is not coordinated with the cobalt nucleus (base-off cobalamin). An additional, variable component of the EPR spectrum of the inactive enzyme has the characteristics of a cob(III)alamin-superoxide complex. Previous work by others [Taylor, R.T., & Weissbach, H. (1969) Arch. Biochem. Biophys. 129, 745-766. Fujii, K., & Huennekens, F.M. (1979) in Biochemical Aspects of Nutrition (Yagi, K., Ed.) pp 173-183, Japan Scientific Societies, Tokyo] has demonstrated that the enzyme can be activated by reductive methylation using adenosylmethionine as the methyl donor. We present data indicating that the conversion of inactive to methylated enzyme is correlated with the disappearance of the EPR spectrum as expected for the conversion of paramagnetic cob(II)alamin to diamagnetic methylcobalamin. When the methyl group is transferred from the methylated enzyme to homocysteine under aerobic conditions, cob(II)alamin/cob(III)alamin-superoxide enzyme is regenerated as indicated by the return of the visible absorbance properties of the initially isolated enzyme and partial return of the EPR spectrum. Our enzyme preparations contain copper in approximately 1:1 stoichiometry with cobalt as determined by atomic absorption spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)

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Photoaffinity labeling of methylenetetrahydrofolate reductase with 8-azido-S-adenosylmethionine.

Cited by 55 publications

References 8 publications

Assignment of enzymic function to specific protein regions of cobalamin-dependent methionine synthase from Escherichia coli

Assignment of enzymic function to specific protein regions of cobalamin-dependent methionine synthase from Escherichia coli

Structures of NADH and CH₃-H₄Folate Complexes of Escherichia coli Methylenetetrahydrofolate Reductase Reveal a Spartan Strategy for a Ping-Pong Reaction^,

Cobalamin-dependent methionine synthase from Escherichia coli B: electron paramagnetic resonance spectra of the inactive form and the active methylated form of the enzyme

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Photoaffinity labeling of methylenetetrahydrofolate reductase with 8-azido-S-adenosylmethionine.

Cited by 55 publications

References 8 publications

Assignment of enzymic function to specific protein regions of cobalamin-dependent methionine synthase from Escherichia coli

Assignment of enzymic function to specific protein regions of cobalamin-dependent methionine synthase from Escherichia coli

Structures of NADH and CH3-H4Folate Complexes of Escherichia coli Methylenetetrahydrofolate Reductase Reveal a Spartan Strategy for a Ping-Pong Reaction,

Cobalamin-dependent methionine synthase from Escherichia coli B: electron paramagnetic resonance spectra of the inactive form and the active methylated form of the enzyme

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Structures of NADH and CH₃-H₄Folate Complexes of Escherichia coli Methylenetetrahydrofolate Reductase Reveal a Spartan Strategy for a Ping-Pong Reaction^,