New insights into inhibitor design from the crystal structure and NMR studies of Escherichia coli GAR transformylase in complex with β-GAR and 10- formyl-5,8,10-trideazafolic acid

Samantha E. Greasley, Mason M. Yamashita, Hui Cai, Stephen Benkovic, Dale L. Boger, Ian A. Wilson

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

The crystal structure of Escherichia coli GAR Tfase at 2.1 A resolution in complex with 10-formyl-5,8,10-trideazafolic acid (10-formyl-TDAF, K(i) = 260 nM), an inhibitor designed to form an enzyme-assembled multisubstrate adduct with the substrate, β-GAR, was studied to determine the exact nature of its inhibitory properties. Rather than forming the expected covalent adduct, the folate inhibitor binds as the hydrated aldehyde (gem-diol) in the enzyme active site, in a manner that mimics the tetrahedral intermediate of the formyl transfer reaction. In this hydrated form, the inhibitor not only provides unexpected insights into the catalytic mechanism but also explains the 10-fold difference in inhibitor potency between 10-formyl-TDAF and the corresponding alcohol, and a further 10-fold difference for inhibitors that lack the alcohol. The presence of the hydrated aldehyde was confirmed in solution by 13C-1H NMR spectroscopy of the ternary GAR Tfase-β-GAR-10- formyl-TDAF complex using the 13C-labeled 10-formyl-TDAF. This insight into the behavior of the inhibitor, which is analogous to protease or transaminase inhibitors, provides a novel and previously unrecognized basis for the design of more potent inhibitors of the folate-dependent formyl transfer enzymes of the purine biosynthetic pathway and development of anti-neoplastic agents.

Original languageEnglish (US)
Pages (from-to)16783-16793
Number of pages11
JournalBiochemistry
Volume38
Issue number51
DOIs
StatePublished - Dec 21 1999

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Phosphoribosylglycinamide Formyltransferase
Escherichia coli
Crystal structure
Nuclear magnetic resonance
Folic Acid
Aldehydes
Enzymes
Alcohols
Gems
Biosynthetic Pathways
Transaminases
Nuclear magnetic resonance spectroscopy
Catalytic Domain
Peptide Hydrolases
Magnetic Resonance Spectroscopy
10-formyl-5,8-10-trideazafolic acid
Substrates

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Greasley, Samantha E. ; Yamashita, Mason M. ; Cai, Hui ; Benkovic, Stephen ; Boger, Dale L. ; Wilson, Ian A. / New insights into inhibitor design from the crystal structure and NMR studies of Escherichia coli GAR transformylase in complex with β-GAR and 10- formyl-5,8,10-trideazafolic acid. In: Biochemistry. 1999 ; Vol. 38, No. 51. pp. 16783-16793.
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abstract = "The crystal structure of Escherichia coli GAR Tfase at 2.1 A resolution in complex with 10-formyl-5,8,10-trideazafolic acid (10-formyl-TDAF, K(i) = 260 nM), an inhibitor designed to form an enzyme-assembled multisubstrate adduct with the substrate, β-GAR, was studied to determine the exact nature of its inhibitory properties. Rather than forming the expected covalent adduct, the folate inhibitor binds as the hydrated aldehyde (gem-diol) in the enzyme active site, in a manner that mimics the tetrahedral intermediate of the formyl transfer reaction. In this hydrated form, the inhibitor not only provides unexpected insights into the catalytic mechanism but also explains the 10-fold difference in inhibitor potency between 10-formyl-TDAF and the corresponding alcohol, and a further 10-fold difference for inhibitors that lack the alcohol. The presence of the hydrated aldehyde was confirmed in solution by 13C-1H NMR spectroscopy of the ternary GAR Tfase-β-GAR-10- formyl-TDAF complex using the 13C-labeled 10-formyl-TDAF. This insight into the behavior of the inhibitor, which is analogous to protease or transaminase inhibitors, provides a novel and previously unrecognized basis for the design of more potent inhibitors of the folate-dependent formyl transfer enzymes of the purine biosynthetic pathway and development of anti-neoplastic agents.",
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New insights into inhibitor design from the crystal structure and NMR studies of Escherichia coli GAR transformylase in complex with β-GAR and 10- formyl-5,8,10-trideazafolic acid. / Greasley, Samantha E.; Yamashita, Mason M.; Cai, Hui; Benkovic, Stephen; Boger, Dale L.; Wilson, Ian A.

In: Biochemistry, Vol. 38, No. 51, 21.12.1999, p. 16783-16793.

Research output: Contribution to journalArticle

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T1 - New insights into inhibitor design from the crystal structure and NMR studies of Escherichia coli GAR transformylase in complex with β-GAR and 10- formyl-5,8,10-trideazafolic acid

AU - Greasley, Samantha E.

AU - Yamashita, Mason M.

AU - Cai, Hui

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AU - Boger, Dale L.

AU - Wilson, Ian A.

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