New Insights into Inhibitor Design from the Crystal Structure and NMR Studies ofEscherichia coliGAR Transformylase in Complex with β-GAR and 10-Formyl-5,8,10-trideazafolic Acid^,

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.

On the Role of Conserved Histidine 106 in 10-Formyltetrahydrofolate Dehydrogenase Catalysis

“…An aspartate/histidine pair is found as a catalytic dyad or part of catalytic triad in the active site of a number of enzymes including serine proteases (16 -18), serine carboxypeptidase (19), phospholipase A 2 (20), dienelactone hydrolase (21,22), ribonuclease A (23), and a variety of zinc-dependent enzymes (24). From the group of 10-formyl-THF-converting enzymes, the GART mechanism and structure have been studied in more detail (15,(25)(26)(27) including resolution of several crystal structures of the enzyme (28 -31). It was shown that replacement of the conserved aspartate with asparagine resulted in catalytically inactive enzyme (15).…”

Crystal Structure and Mechanism of theEscherichia coliArnA (PmrI) Transformylase Domain. An Enzyme for Lipid A Modification with 4-Amino-4-deoxy-l-arabinose and Polymyxin Resistance,

“…The structure of GARF has been solved with various N-10-formyltetrahydrofolate analogues bound to the active site including the formylated analogue 10-formyl-5,8,10-tride-azafolic acid (NHS) (47). On the basis of the sequence and structure similarities between GARF and ArnA_TF described above, we modeled NHS in the active site of ArnA_TF using as a guide the position of NHS in GARF [PDB_ID 1C2T (47)].…”

“…This water molecule makes hydrogen bonds with O δ 1 of D 140 (2.9 Å), N δ 2 of N 102 (2.9 Å), and N δ 1 of H 104 (2.7 Å), the three residues implicated in the formyl transfer reaction ( Figure 4C). The main chain atoms of G 113 hydrogen bond with both the hydroxyl of the hydrated aldehyde of NHS [that represents where the primary amino group from the second substrate, UDP-Ara4N, would be (47)] and the side chain oxygen of the catalytically important D 140 bringing them together. Therefore, G 113 positions the amino group of UDP-Ara4N and the catalytic D 140 in a favorable conformation for catalysis.…”

Involvement of a transformylase enzyme in siderophore synthesis in Pseudomonas aeruginosa The GenBank accession number for the sequence reported in this paper is U07359.

“…GART is involved in the de novo synthesis of purines, catalysing the formylation of glycinamide ribonucleotide to formylglycinamide ribonucleotide in conjunction with the cofactor N"!-formyltetrahydrofolate (Buchanan & Hartman, 1959). Several of the residues identified as being important for the function of the E. coli version of GART (Almassy et al, 1992 ;Chen et al, 1992 ;Warren et al, 1996 ;Shim & Benkovic, 1999 ;Greasley et al, 1999) are present in the PvdF sequence (Fig. 3).…”

“…Positions where identical or similar residues are present in at least five of the sequences are highlighted, with identical residues shaded black and similar residues (A and G ; D and E ; F, W and Y ; I, L, M and V ; N and Q ; S and T ; H, R and K) shaded grey. Residues that are known to be involved in catalysis by GART from E. coli (Warren et al, 1996 ;Shim & Benkovic, 1999) are marked with an asterisk, and those that form a hydrophobic pocket and are likely to interact with formyl tetrahydrofolate (Almassy et al, 1992 ;Chen et al, 1992 ;Greasley et al, 1999) are marked with a dagger.…”