pubs.acs.org/Biochemistry Published on Web 08/26/2010 r 2010 American Chemical Society 9078 Biochemistry 2010, 49, 9078–9088 DOI: 10.1021/bi1007222 The Tail Wagging the Dog: Insights into Catalysis in R67 Dihydrofolate Reductase † Ganesh Kamath, ‡ Elizabeth E. Howell, § and Pratul K. Agarwal* ,‡ ‡ Computer Science and Mathematics Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, and § Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996 Received May 6, 2010; Revised Manuscript Received August 26, 2010 ABSTRACT: Plasmid-encoded R67 dihydrofolate reductase (DHFR) catalyzes a hydride transfer reaction between substrate dihydrofolate (DHF) and its cofactor, nicotinamide adenine dinucleotide phosphate (NADPH). R67 DHFR is a homotetramer that exhibits numerous characteristics of a primitive enzyme, including promiscuity in binding of substrate and cofactor, formation of nonproductive complexes, and the absence of a conserved acid in its active site. Furthermore, R67’s active site is a pore, which is mostly accessible by bulk solvent. This study uses a computational approach to characterize the mechanism of hydride transfer. Not surprisingly, NADPH remains fixed in one-half of the active site pore using numerous interactions with R67. Also, stacking between the nicotinamide ring of the cofactor and the pteridine ring of the substrate, DHF, at the hourglass center of the pore, holds the reactants in place. However, large movements of the p-aminoben- zoylglutamate tail of DHF occur in the other half of the pore because of ion pair switching between symmetry- related K32 residues from two subunits. This computational result is supported by experimental results that the loss of these ion pair interactions (located >13 A ˚ from the center of the pore) by addition of salt or in asymmetric K32M mutants leads to altered enzyme kinetics [Hicks, S. N., et al. (2003) Biochemistry 42, 10569-10578; Hicks, S. N., et al. (2004) J. Biol. Chem. 279, 46995-47002]. The tail movement at the edge of the active site, coupled with the fixed position of the pteridine ring in the center of the pore, leads to puckering of the pteridine ring and promotes formation of the transition state. Flexibility coupled to R67 function is unusual as it contrasts with the paradigm that enzymes use increased rigidity to facilitate attainment of their transition states. A comparison with chromosomal DHFR indicates a number of similarities, including puckering of the nicotinamide ring and changes in the DHF tail angle, accomplished by different elements of the dissimilar protein folds. R-Plasmid-encoded dihydrofolate reductase (R67 DHFR) 1 catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofo- late (THF) using nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. While the reaction is the same as that catalyzed by the chromosomally encoded DHFR in Escherichia coli (EcDHFR) and other organisms, R67 shows neither se- quence nor structural homology with chromosomal DHFRs (1-4). This type II DHFR was discovered because of its ability to confer trimethoprim resistance upon host bacteria (5). Each R67 DHFR monomer is 78 amino acids long, and the subunits assemble into a homotetramer possessing 222 symmetry (see Figure 1). A single active site pore with a volume of 3626 A ˚ 3 traverses the length of the molecule. R67 DHFR possesses a SH3 domain-like fold, while EcDHFR displays a Rossmann fold, the latter being a characteristic feature of dinucleotide binding proteins (6). R67 is an unusual enzyme as it uses symmetry- related sites to bind various combinations of ligands, including two substrate (DHF) molecules or two cofactor (NADPH) molecules or the catalytically relevant combination of one DHF molecule and one NADPH molecule (7). R67 DHFR has been described as a primitive enzyme because of several features, including an unusual active site pore that is mostly accessible by bulk water except at the hourglass center, no conserved acid or base in the pore, formation of nonproductive 2DHF or 2NADPH complexes, and a low catalytic efficiency (∼3  10 5 s -1 M -1 ) compared to that of EcDHFR (1). More- over, this enzyme behaves in a manner different from the conventional paradigm of enzyme catalysis in a number of ways. First, the binding-site promiscuity for different ligands in R67 deviates from the lock-and-key hypothesis, as DHF and NADPH share symmetry-related sites; R-NADPH can be used as cofactor (8), and novobiocin and congo red are inhibitors [with K i values of 70 and 2 μM, respectively (9, 10)]. Neither of the latter two ligands resembles the substrate and/or the cofactor. Second, a widely accepted view of enzyme catalysis suggests that an increase in rigidity in the active site or enthalpy driven binding of the transition state facilitates catalysis (11-15). In other words, loss of translational and rotational motion in a well- evolved active site leads to the correct positioning of enzyme and reactants, thus facilitating the chemical step of the reaction. † The contributions of G.K. and P.K.A. were supported by Oak Ridge National Laboratory’s Laboratory Directed Research and Develop- ment funds and the allocation of computing time by the National Center for Computational Sciences (BIP003). The contribution of E.E.H. was supported by National Science Foundation Grant MCB-0817827. *To whom correspondence should be addressed: Oak Ridge National Laboratory, P.O. Box 2008, MS 6016, Oak Ridge, TN 37831. Phone: (865) 574-7184. Fax: (865) 576-5491. E-mail: agarwalpk@ornl.gov. 1 Abbreviations: C A , acceptor carbon; C D , donor carbon; DHF, dihydrofolate; DHFR, dihydrofolate reductase; EcDHFR, E. coli dihydrofolate reductase; EVB, empirical valence bond; K32M, muta- tion in which lysine 32 is replaced with methionine; p-ABG, p-amino- benzoylglutamate; NADPH, reduced form of nicotinamide adenine dinucleotide phosphate; NADP þ , oxidized form of nicotinamide ade- nine dinucleotide phosphate; R67 DHFR, plasmid-encoded type II dihydrofolate reductase; MD, molecular dynamics.