Identification of endogenous ligands bound to bacterially expressed human and E. coli dihydrofolate reductase by 2D NMR
► NMR data show that endogenous ligands copurify with DHFR expressed in bacterial cells. ► Human DHFR is preferentially bound to NADP, and Escherichia coli DHFR is bound to THF. ► An E. coli DHFR mutant switches binding specificities to resemble the human enzyme. ► We describe purification schemes t...
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| Published in | FEBS letters Vol. 585; no. 22; pp. 3528 - 3532 |
|---|---|
| Main Authors | , , , |
| Format | Journal Article |
| Language | English |
| Published |
England
Elsevier B.V
16.11.2011
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| Subjects | |
| Online Access | Get full text |
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| Abstract | ► NMR data show that endogenous ligands copurify with DHFR expressed in bacterial cells. ► Human DHFR is preferentially bound to NADP, and
Escherichia coli DHFR is bound to THF. ► An
E. coli DHFR mutant switches binding specificities to resemble the human enzyme. ► We describe purification schemes to obtain DHFR samples with only desired ligands.
Dihydrofolate reductase (DHFR) is a well-studied drug target and a paradigm for understanding enzyme catalysis. Preparation of pure DHFR samples, in defined ligand-bound states, is a prerequisite for in vitro studies and drug discovery efforts. We use NMR spectroscopy to monitor ligand content of human and
Escherichia coli DHFR (ecDHFR), which bind different co-purifying ligands during expression in bacteria. An alternate purification strategy yields highly pure DHFR complexes, containing only the desired ligands, in the quantities required for structural studies. Interestingly, ecDHFR is bound to endogenous THF while human DHFR is bound to NADP. Consistent with these findings, a designed “humanized” mutant of ecDHFR switches binding specificity in the cell. |
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| AbstractList | Dihydrofolate reductase (DHFR) is a well-studied drug target and a paradigm for understanding enzyme catalysis. Preparation of pure DHFR samples, in defined ligand-bound states, is a prerequisite for in vitro studies and drug discovery efforts. We use NMR spectroscopy to monitor ligand content of human and Escherichia coli DHFR (ecDHFR), which bind different co-purifying ligands during expression in bacteria. An alternate purification strategy yields highly pure DHFR complexes, containing only the desired ligands, in the quantities required for structural studies. Interestingly, ecDHFR is bound to endogenous THF while human DHFR is bound to NADP. Consistent with these findings, a designed "humanized" mutant of ecDHFR switches binding specificity in the cell. Dihydrofolate reductase (DHFR) is a well-studied drug target and a paradigm for understanding enzyme catalysis. Preparation of pure DHFR samples, in defined ligand-bound states, is a prerequisite for in vitro studies and drug discovery efforts. We use NMR spectroscopy to monitor ligand content of human and E. coli DHFR (ecDHFR), which bind different co-purifying ligands during expression in bacteria. An alternate purification strategy yields highly pure DHFR complexes, containing only the desired ligands, in the quantities required for structural studies. Interestingly, ec DHFR is bound to endogenous THF while human DHFR is bound to NADP. Consistent with these findings, a designed “humanized” mutant of ec DHFR switches binding specificity in the cell. Dihydrofolate reductase (DHFR) is a well‐studied drug target and a paradigm for understanding enzyme catalysis. Preparation of pure DHFR samples, in defined ligand‐bound states, is a prerequisite for in vitro studies and drug discovery efforts. We use NMR spectroscopy to monitor ligand content of human and Escherichia coli DHFR (ecDHFR), which bind different co‐purifying ligands during expression in bacteria. An alternate purification strategy yields highly pure DHFR complexes, containing only the desired ligands, in the quantities required for structural studies. Interestingly, ecDHFR is bound to endogenous THF while human DHFR is bound to NADP. Consistent with these findings, a designed “humanized” mutant of ecDHFR switches binding specificity in the cell. ► NMR data show that endogenous ligands copurify with DHFR expressed in bacterial cells. ► Human DHFR is preferentially bound to NADP, and Escherichia coli DHFR is bound to THF. ► An E. coli DHFR mutant switches binding specificities to resemble the human enzyme. ► We describe purification schemes to obtain DHFR samples with only desired ligands. ► NMR data show that endogenous ligands copurify with DHFR expressed in bacterial cells. ► Human DHFR is preferentially bound to NADP, and Escherichia coli DHFR is bound to THF. ► An E. coli DHFR mutant switches binding specificities to resemble the human enzyme. ► We describe purification schemes to obtain DHFR samples with only desired ligands. Dihydrofolate reductase (DHFR) is a well-studied drug target and a paradigm for understanding enzyme catalysis. Preparation of pure DHFR samples, in defined ligand-bound states, is a prerequisite for in vitro studies and drug discovery efforts. We use NMR spectroscopy to monitor ligand content of human and Escherichia coli DHFR (ecDHFR), which bind different co-purifying ligands during expression in bacteria. An alternate purification strategy yields highly pure DHFR complexes, containing only the desired ligands, in the quantities required for structural studies. Interestingly, ecDHFR is bound to endogenous THF while human DHFR is bound to NADP. Consistent with these findings, a designed “humanized” mutant of ecDHFR switches binding specificity in the cell. |
| Author | Bhabha, Gira Martinez-Yamout, Maria A. Tuttle, Lisa Wright, Peter E. |
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| Copyright | 2011 Federation of European Biochemical Societies FEBS Letters 585 (2011) 1873-3468 © 2015 Federation of European Biochemical Societies Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 2011 |
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| Keywords | NMR Purification Dihydrofolate reductase Expression Ligand |
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| Snippet | ► NMR data show that endogenous ligands copurify with DHFR expressed in bacterial cells. ► Human DHFR is preferentially bound to NADP, and
Escherichia coli... Dihydrofolate reductase (DHFR) is a well‐studied drug target and a paradigm for understanding enzyme catalysis. Preparation of pure DHFR samples, in defined... Dihydrofolate reductase (DHFR) is a well-studied drug target and a paradigm for understanding enzyme catalysis. Preparation of pure DHFR samples, in defined... |
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| SubjectTerms | Binding Sites Dihydrofolate reductase Escherichia coli Escherichia coli - enzymology Escherichia coli - metabolism Expression Folic Acid - metabolism Humans Ligand Ligands Magnetic Resonance Spectroscopy Models, Molecular NADP - chemistry NADP - metabolism NMR Purification Tetrahydrofolate Dehydrogenase - chemistry Tetrahydrofolate Dehydrogenase - genetics Tetrahydrofolate Dehydrogenase - metabolism Tetrahydrofolates - metabolism |
| Title | Identification of endogenous ligands bound to bacterially expressed human and E. coli dihydrofolate reductase by 2D NMR |
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