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

Greasley SE, Yamashita MM, Cai H, Benkovic SJ, Boger DL, Wilson IA
Biochemistry 38 16783-93 (1999)
Cited: 23 times
EuropePMC logo PMID: 10606510

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, beta-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 (13)C-(1)H NMR spectroscopy of the ternary GAR Tfase-beta-GAR-10-formyl-TDAF complex using the (13)C-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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  1. Structural biology of the purine biosynthetic pathway. Zhang Y, Morar M, Ealick SE. Cell Mol Life Sci 65 3699-3724 (2008)

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  1. Crystal structure and mechanism of the Escherichia coli ArnA (PmrI) transformylase domain. An enzyme for lipid A modification with 4-amino-4-deoxy-L-arabinose and polymyxin resistance. Gatzeva-Topalova PZ, May AP, Sousa MC. Biochemistry 44 5328-5338 (2005)
  2. Design, synthesis and biological evaluation of 10-CF3CO-DDACTHF analogues and derivatives as inhibitors of GAR Tfase and the de novo purine biosynthetic pathway. Desharnais J, Hwang I, Zhang Y, Tavassoli A, Baboval J, Benkovic SJ, Wilson IA, Boger DL. Bioorg Med Chem 11 4511-4521 (2003)
  3. Modular organization of FDH: Exploring the basis of hydrolase catalysis. Reuland SN, Vlasov AP, Krupenko SA. Protein Sci 15 1076-1084 (2006)
  4. 10-Formyl-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic acid (10-formyl-DDACTHF): a potent cytotoxic agent acting by selective inhibition of human GAR Tfase and the de novo purine biosynthetic pathway. Marsilje TH, Labroli MA, Hedrick MP, Jin Q, Desharnais J, Baker SJ, Gooljarsingh LT, Ramcharan J, Tavassoli A, Zhang Y, Wilson IA, Beardsley GP, Benkovic SJ, Boger DL. Bioorg Med Chem 10 2739-2749 (2002)
  5. Design, synthesis, and biological evaluation of simplified alpha-keto heterocycle, trifluoromethyl ketone, and formyl substituted folate analogues as potential inhibitors of GAR transformylase and AICAR transformylase. Marsilje TH, Hedrick MP, Desharnais J, Tavassoli A, Zhang Y, Wilson IA, Benkovic SJ, Boger DL. Bioorg Med Chem 11 4487-4501 (2003)
  6. Native-state conformational dynamics of GART: a regulatory pH-dependent coil-helix transition examined by electrostatic calculations. Morikis D, Elcock AH, Jennings PA, McCammon JA. Protein Sci 10 2363-2378 (2001)
  7. Percentile-based spread: a more accurate way to compare crystallographic models. Pozharski E. Acta Crystallogr D Biol Crystallogr 66 970-978 (2010)
  8. Proton transfer dynamics of GART: the pH-dependent catalytic mechanism examined by electrostatic calculations. Morikis D, Elcock AH, Jennings PA, McCammon JA. Protein Sci 10 2379-2392 (2001)
  9. Structures of glycinamide ribonucleotide transformylase (PurN) from Mycobacterium tuberculosis reveal a novel dimer with relevance to drug discovery. Zhang Z, Caradoc-Davies TT, Dickson JM, Baker EN, Squire CJ. J Mol Biol 389 722-733 (2009)
  10. Synthesis and biological evaluation of N-[4-[5-(2,4-diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-2-(2,2,2-trifluoroacetyl)pentyl]benzoyl]-L-glutamic acid as a potential inhibitor of GAR Tfase and the de novo purine biosynthetic pathway. Cheng H, Hwang I, Chong Y, Tavassoli A, Webb ME, Zhang Y, Wilson IA, Benkovic SJ, Boger DL. Bioorg Med Chem 13 3593-3599 (2005)
  11. Biological and structural evaluation of 10R- and 10S-methylthio-DDACTHF reveals a new role for sulfur in inhibition of glycinamide ribonucleotide transformylase. Connelly S, DeMartino JK, Boger DL, Wilson IA. Biochemistry 52 5133-5144 (2013)
  12. 10-(2-benzoxazolcarbonyl)-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic acid: a potential inhibitor of GAR transformylase and AICAR transformylase. Marsilje TH, Hedrick MP, Desharnais J, Capps K, Tavassoli A, Zhang Y, Wilson IA, Benkovic SJ, Boger DL. Bioorg Med Chem 11 4503-4509 (2003)
  13. Multisubstrate adduct inhibitors: drug design and biological tools. Le Calvez PB, Scott CJ, Migaud ME. J Enzyme Inhib Med Chem 24 1291-1318 (2009)
  14. Structures and reaction mechanisms of the two related enzymes, PurN and PurU. Sampei G, Kanagawa M, Baba S, Shimasaki T, Taka H, Mitsui S, Fujiwara S, Yanagida Y, Kusano M, Suzuki S, Terao K, Kawai H, Fukai Y, Nakagawa N, Ebihara A, Kuramitsu S, Yokoyama S, Kawai G. J Biochem 154 569-579 (2013)
  15. The pH dependence of stability of the activation helix and the catalytic site of GART. Morikis D, Elcock AH, Jennings PA, McCammon JA. Biophys Chem 105 279-291 (2003)
  16. Asymmetric synthesis of inhibitors of glycinamide ribonucleotide transformylase. DeMartino JK, Hwang I, Connelly S, Wilson IA, Boger DL. J Med Chem 51 5441-5448 (2008)
  17. Design, synthesis, and biological evaluation of 10-methanesulfonyl-DDACTHF, 10-methanesulfonyl-5-DACTHF, and 10-methylthio-DDACTHF as potent inhibitors of GAR Tfase and the de novo purine biosynthetic pathway. Cheng H, Chong Y, Hwang I, Tavassoli A, Zhang Y, Wilson IA, Benkovic SJ, Boger DL. Bioorg Med Chem 13 3577-3585 (2005)
  18. Human glycinamide ribonucleotide transformylase: active site mutants as mechanistic probes. Manieri W, Moore ME, Soellner MB, Tsang P, Caperelli CA. Biochemistry 46 156-163 (2007)
  19. Classification of ligand molecules in PDB with graph match-based structural superposition. Shionyu-Mitsuyama C, Hijikata A, Tsuji T, Shirai T. J Struct Funct Genomics 17 135-146 (2016)
  20. A quantum chemical study on the mechanism of glycinamide ribonucleotide transformylase inhibitor: 10-Formyl-5,8,10-trideazafolic acid. Qiao QA, Jin Y, Yang C, Zhang Z, Wang M. Biophys Chem 118 78-83 (2005)
  21. Stereoselective Synthesis of β-Glycinamide Ribonucleotide. Ngu L, Ray D, Watson SS, Beuning PJ, Ondrechen MJ, O'Doherty GA. Molecules 27 2528 (2022)


Related citations provided by authors (2)

  1. A pH Dependent Stabilization of an Active Site Loop Observed from Low and High pH Crystal Structures of Mutant Monomeric Glycinamide Ribonucleotide Transformylase at 1.8-1.9A. Su Y, Yamashita MM, Greasley SE, Mullen CA, Shim JH, Jennings PA, Benkovic SJ, Wilson IA J. Mol. Biol. 281 485-499 (1998)
  2. 10-Formyl-5,8,10-trideazafolic acid (10-formyl-TDAF):a Potent Inhibitor of Glycinamide Ribonucleotide Transformylase.. Boger DL, Haynes N-E, Kitos PA, Ramcharan J, Marolewski AE, Benkovic SJ Bioorg. Med. Chem. 5 1817-1830 (1997)

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