Structural comparison of chromosomal and exogenous dihydrofolate reductase from Staphylococcus aureus in complex with the potent inhibitor trimethoprim

Abstract

Dihydrofolate reductase (DHFR) is the enzyme responsible for the NADPH-dependent reduction of 5,6-dihydrofolate to 5,6,7,8-tetrahydrofolate, an essential cofactor in the synthesis of purines, thymidylate, methionine, and other key metabolites. Because of its importance in multiple cellular functions, DHFR has been the subject of much research targeting the enzyme with anticancer, antibacterial, and antimicrobial agents. Clinically used compounds targeting DHFR include methotrexate for the treatment of cancer and diaminopyrimidines (DAPs) such as trimethoprim (TMP) for the treatment of bacterial infections. DAP inhibitors of DHFR have been used clinically for >30 years and resistance to these agents has become widespread. Methicillin-resistant Staphylococcus aureus (MRSA), the causative agent of many serious nosocomial and community acquired infections, and other gram-positive organisms can show resistance to DAPs through mutation of the chromosomal gene or acquisition of an alternative DHFR termed ''S1 DHFR.'' To develop new therapies for health threats such as MRSA, it is important to understand the molecular basis of DAP resistance. Here, we report the crystal structure of the wild-type chromosomal DHFR from S. aureus in complex with NADPH and TMP. We have also solved the structure of the exogenous, TMP resistant S1 DHFR, apo and in complex with TMP. The structural and thermodynamic data point to important molecular differences between the two enzymes that lead to dramatically reduced affinity of DAPs to S1 DHFR. These differences in enzyme binding affinity translate into reduced antibacterial activity against strains of S. aureus that express S1 DHFR. Proteins 2009; 76:706-717. V V C 2009 Wiley-Liss, Inc.

Nonracemic Antifolates Stereoselectively Recruit Alternate Cofactors and Overcome Resistance in S. aureus

“…However, while this structure of Sa(F98Y):β-NADPH:TMP may be thermodynamically stable in the crystal, there is little evidence that it is kinetically stable in solution, as shown by isothermal calorimetry experiments revealing attenuated binding with or without NADPH 7 and dissociation experiments shown here that reveal a very fast dissolution of the complex between Sa(F98Y) and TMP. Although studies on the catalytic cycle of DHFR have described a classical binding order where NADPH binds prior to DHF 33 , various solution experiments (**ref Heaslet and Polshakov) have shown that the binding of TMP and NADPH can occur in any order, ultimately leading to a stable ternary complex.…”

“…3–6 The F98Y point mutation in DHFR is the pivotal mutation clinically observed to confer high levels of resistance to trimethoprim, 5 primarily resulting in a change in entropy of ligand binding and a loss of synergy, or binding affinity, between the inhibitor and NADPH cofactor. 7 New generations of antifolates that effectively target the mutated forms of DHFR will be critical for prolonging the utility of this class of antibiotics.…”

Localized light-induced protein dimerization in living cells using a photocaged dimerizer

“…Linkers can be added to the para position of the trimethoxyphenyl ring of TMP without interfering with DHFR binding 16 . By contrast, the 2,4-diaminopyrimidine ring of TMP is buried in the DHFR substrate pocket 21 , so we predicted that adding a bulky photocage to this group would effectively block DHFR binding. Neither photocaged TMP nor a photocaged modular chemical inducer of dimerization have been reported.…”

Antibacterial Antifolates: From Development through Resistance to the Next Generation

“…A crystal structure of S1 with TMP revealed six molecules in the asymmetric unit, only three of which were the ternary structure. The other three S1 proteins bound only TMP, indicating the altered interactions of the S1 enzyme with NADPH (Heaslet et al 2009). …”

“…4A). Interestingly, this residue is an isoleucine in S1 DHFR, which is known to contribute to TMP resistance (Heaslet et al 2009). In the Gram-negative enzymes, Phe at position 31 is balanced by less bulky residues on the opposite side of the active site (Fig.…”