Metabolic engineering in yeast demonstrates that S-adenosylmethionine controls flux through the methylenetetrahydrofolate reductase reaction in vivo

Sanja Roje; Sherwin Y Chan; Fatma Kaplan; Rhonda K Raymond; Donald W Horne; Dean R Appling; Andrew D Hanson

doi:10.1074/jbc.M110651200

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Metabolic engineering in yeast demonstrates that S-adenosylmethionine controls flux through the methylenetetrahydrofolate reductase reaction in vivo

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Metabolic engineering in yeast demonstrates that S-adenosylmethionine controls flux through the methylenetetrahydrofolate reductase reaction in vivo

Sanja Roje, Sherwin Y Chan, Fatma Kaplan, Rhonda K Raymond, Donald W Horne, Dean R Appling and Andrew D Hanson

The Journal of biological chemistry, Vol.277(6), pp.4056-4061

02/08/2002

DOI: https://doi.org/10.1074/jbc.M110651200

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https://hdl.handle.net/2376/109587

PMID: 11729203

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Abstract

Amino Acid Sequence

Arabidopsis - enzymology

Oxidoreductases Acting on CH-NH Group Donors - chemistry

Molecular Sequence Data

Oxidoreductases Acting on CH-NH Group Donors - genetics

Methylenetetrahydrofolate Reductase (NADPH2)

Oxidoreductases Acting on CH-NH Group Donors - metabolism

DNA Primers

Genetic Complementation Test

Arabidopsis - genetics

Saccharomyces cerevisiae - metabolism

Base Sequence

Folic Acid - metabolism

Nuclear Magnetic Resonance, Biomolecular

Kinetics

Mutation

NADP - metabolism

S-Adenosylmethionine - metabolism

One-carbon flux into methionine and S-adenosylmethionine (AdoMet) is thought to be controlled at the methylenetetrahydrofolate reductase (MTHFR) step. Mammalian MTHFRs are inhibited by AdoMet in vitro, and it has been proposed that methyl group biogenesis is regulated in vivo by this feedback loop. In this work, we used metabolic engineering in the yeast Saccharomyces cerevisiae to test this hypothesis. Like mammalian MTHFRs, the yeast MTHFR encoded by the MET13 gene is NADPH-dependent and is inhibited by AdoMet in vitro. This contrasts with plant MTHFRs, which are NADH-dependent and AdoMet-insensitive. To manipulate flux through the MTHFR reaction in yeast, the chromosomal copy of MET13 was replaced by an Arabidopsis MTHFR cDNA (AtMTHFR-1) or by a chimeric sequence (Chimera-1) comprising the yeast N-terminal domain and the AtMTHFR-1 C-terminal domain. Chimera-1 used both NADH and NADPH and was insensitive to AdoMet, supporting the view that the C-terminal domain is responsible for AdoMet inhibition. Engineered yeast expressing Chimera-1 accumulated 140-fold more AdoMet and 7-fold more methionine than did the wild-type and grew normally. Yeast expressing AtMTHFR-1 accumulated 8-fold more AdoMet. This is the first in vivo evidence that the AdoMet sensitivity and pyridine nucleotide preference of MTHFR control methylneogenesis. (13)C labeling data indicated that glycine cleavage becomes a more prominent source of one-carbon units when Chimera-1 is expressed. Possibly related to this shift in one-carbon fluxes, total folate levels are doubled in yeast cells expressing Chimera-1.

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Title: Metabolic engineering in yeast demonstrates that S-adenosylmethionine controls flux through the methylenetetrahydrofolate reductase reaction in vivo
Creators: Sanja Roje - Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611, USA
Sherwin Y Chan
Fatma Kaplan
Rhonda K Raymond
Donald W Horne
Dean R Appling
Andrew D Hanson
Publication Details: The Journal of biological chemistry, Vol.277(6), pp.4056-4061
Academic Unit: Biological Chemistry, Institute of
Publisher: United States
Grant note: DK32189 / NIDDK NIH HHS DK26657 / NIDDK NIH HHS
Identifiers: 99900547126801842
Language: English
Resource Type: Journal article