Construction of an altered proton donation mechanism in Escherichia coli dihydrofolate reductase

Howell, Elizabeth E.; Warren, Mark S.; Booth, Carol L. J.; Villafranca, J. E.; Kraut, Joseph

doi:10.1021/bi00400a015

Cited by 47 publications

(72 citation statements)

References 48 publications

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“…In contrast, the F137S mutant did not show a large increase in its methotrexate dissociation constant as compared to the wild-type enzyme (0.13 nM vs. 0.068 nM; ref. 14), which is consistent with the observation that the ionic and hydrogen bonding interactions between Asp-27 and methotrexate are identical in these two structures. In the case of the D27S/ F137S double mutant, a methotrexate dissociation constant of 281 nM is to be compared with 55 nM for the D27S single mutant (14).…”

Section: Resultssupporting

confidence: 90%

“…14), which is consistent with the observation that the ionic and hydrogen bonding interactions between Asp-27 and methotrexate are identical in these two structures. In the case of the D27S/ F137S double mutant, a methotrexate dissociation constant of 281 nM is to be compared with 55 nM for the D27S single mutant (14). Although the refined crystal structures show that the positions of both Ser-27 and methotrexate are essentially identical in D27S and D27S/F137S, there are significant structural perturbations in the aB helix, as discussed earlier; an important point is that some side chains ofthe aB helix are in contact with the bound methotrexate.…”

Section: Resultssupporting

confidence: 90%

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Long-range structural effects in a second-site revertant of a mutant dihydrofolate reductase.

Brown

Howell

Kraut

1993

Proc. Natl. Acad. Sci. U.S.A.

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Section: Resultssupporting

confidence: 90%

Section: Resultssupporting

confidence: 90%

Long-range structural effects in a second-site revertant of a mutant dihydrofolate reductase.

Brown

Howell

Kraut

1993

Proc. Natl. Acad. Sci. U.S.A.

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“…Steady State Kinetics-Steady state kinetic data were obtained using a PerkinElmer Life Sciences 35 spectrophotometer interfaced with an IBM personal computer as described previously (19). MTA (MTA buffer: 100 mM Tris, 50 mM MES, 50 mM acetic acid polybuffer plus 1 mM ␤-mercaptoethanol) polybuffer was used.…”

Section: Methodsmentioning

confidence: 99%

A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

Chopra

Dooling

Horner

et al. 2008

Journal of Biological Chemistry

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R67 dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate (DHF) to tetrahydrofolate using NADPH as a cofactor. This enzyme is a homotetramer possessing 222 symmetry, and a single active site pore traverses the length of the protein. A promiscuous binding surface can accommodate either DHF or NADPH, thus two nonproductive complexes can form (2NADPH or 2DHF) as well as a productive complex (NADPH⅐DHF). The role of water in binding was monitored using a number of different osmolytes. From isothermal titration calorimetry (ITC) studies, binding of NADPH is accompanied by the net release of 38 water molecules. In contrast, from both steady state kinetics and ITC studies, binding of DHF is accompanied by the net uptake of water. Although different osmolytes have similar effects on NADPH binding, variable results are observed when DHF binding is probed. Sensitivity to water activity can also be probed by an in vivo selection using the antibacterial drug, trimethoprim, where the water content of the media is decreased by increasing concentrations of sorbitol. The ability of wild type and mutant clones of R67 DHFR to allow host Escherichia coli to grow in the presence of trimethoprim plus added sorbitol parallels the catalytic efficiency of the DHFR clones, indicating water content strongly correlates with the in vivo function of R67 DHFR.R67 dihydrofolate reductase, a type II DHFR, 2 catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate. The gene for this enzyme is carried by an R-plasmid, and its presence confers resistance to the antibiotic drug, trimethoprim (TMP). Trimethoprim is a competitive inhibitor of chromosomal DHFR with a picomolar K i (1). Although R67 DHFR is not a good catalyst, it is not inhibited by TMP very well, and thus it allows cell growth in the presence of this antibacterial drug (2, 3). R67 DHFR shares no homology in sequence or structure with chromosomal DHFR and has been proposed to be a good model of a primitive enzyme (4).R67 DHFR is a homotetramer with a single active site pore; the overall structure possesses 222 symmetry as seen in Fig. 1 (5). The symmetry of the active site results in overlapping binding sites for substrate, DHF, and cofactor, NADPH. This can be observed as R67 DHFR binds a total of two ligands as follows: either two NADPH molecules or two folate/DHF molecules or one NADPH plus one folate/DHF molecule (6). The first two complexes are dead-end (binary) complexes, whereas the third is the productive ternary complex. Because of the 222 symmetry, binding to either ligand is unlikely to be optimal.The active site pore of R67 DHFR is unusual in its hourglass shape as well as its large size (2938 Å 3 for apoR67 DHFR with hydrogens added, calculated by CASTp (4, 7)). Because of this large volume, DHF and NADPH cannot occupy all the space in the pore and must use water to mediate some contacts with the protein.How do substrate and cofactor bind to R67 DHFR? From NMR, crystallography, and docking studies, the pteridine ring of DHF/fo...

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“…Steady-state Kinetics with Mutants-The steady-state kinetic behavior of each mutant was monitored as previously described (19). Experiments were performed at 30°C in either 50 mM MES plus 100 mM Tris, plus 50 mM acetic acid (MTH buffer) polybuffer (20) or TE buffer (10 mM Tris, 1 mM EDTA, pH 7) in the presence of various concentrations of NaCl to adjust the ionic strength.…”

Section: Methodsmentioning

confidence: 99%

Role of Lys-32 Residues in R67 Dihydrofolate Reductase Probed by Asymmetric Mutations

Hicks

Smiley

Stinnett

et al. 2004

Journal of Biological Chemistry

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R67 dihydrofolate reductase (R67 DHFR) is a novel protein encoded by an R-plasmid that confers resistance to the antibiotic, trimethoprim. This homotetrameric enzyme possesses 222 symmetry, which imposes numerous constraints on the single active site pore, including a "one-site-fits-both" strategy for binding its ligands, dihydrofolate (DHF) and NADPH. Previous studies uncovered salt effects on binding and catalysis (Hicks, S. N., Smiley, R. D., Hamilton, J. B., and Howell, E. E. (2003) Biochemistry 42, 10569 -10578), however the one or more residues that participate in ionic contacts with the negatively charged tail of DHF as well as the phosphate groups in NADPH were not identified. Several studies predict that Lys-32 residues were involved, however mutations at this residue destabilize the R67 DHFR homotetramer. To study the role of Lys-32 in binding and catalysis, asymmetric K32M mutations have been utilized. To create asymmetry, individual mutations were added to a tandem array of four in-frame gene copies. These studies show one K32M mutation is tolerated quite well, whereas addition of two mutations has variable effects. Two double mutants, K32M:1؉2 and K32M: 1؉4, which place the mutations on opposite sides of the pore, reduce k cat . However a third double mutant, K32M: 1؉3, that places two mutations on the same half pore, enhances k cat 4-to 5-fold compared with the parent enzyme, albeit at the expense of weaker binding of ligands. Because the k cat /K m values for this double mutant series are similar, these mutations appear to have uncovered some degree of non-productive binding. This non-productive binding mode likely arises from formation of an ionic interaction that must be broken to allow access to the transition state. The K32M:1؉3 mutant data suggest this interaction is an ionic interaction between Lys-32 and the charged tail of dihydrofolate. This unusual catalytic scenario arises from the 222 symmetry imposed on the single active site pore. R67 dihydrofolate reductase (R67 DHFR)1 is an R-plasmidencoded enzyme that catalyzes the NADPH-dependent reduction of dihydrofolate (DHF) to tetrahydrofolate. Its presence in bacteria confers resistance to the antibiotic, trimethoprim. This enzyme is not similar in sequence or structure to the chromosomally encoded DHFRs.R67 DHFR is a homotetramer, and the pore that traverses the length of the molecule is the active site. Surprisingly, the structure possesses 222 symmetry (1), which imposes numerous constraints on binding and catalysis. For example, the symmetry requires that for each binding site, there must be three additional, symmetry-related sites. However, solution studies find only two sites can be occupied simultaneously because of steric constraints. The possible binding combinations are two NADPH molecules, or two folate/DHF molecules, or one NADPH plus one folate/DHF molecule (2). Only the latter is productive. Thus binding of neither ligand can be optimized, and a "one-site-fits-both" approach is employed (3, 4). Another constraint arising from ...

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Construction of an altered proton donation mechanism in Escherichia coli dihydrofolate reductase

Cited by 47 publications

References 48 publications

Long-range structural effects in a second-site revertant of a mutant dihydrofolate reductase.

Long-range structural effects in a second-site revertant of a mutant dihydrofolate reductase.

A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

Role of Lys-32 Residues in R67 Dihydrofolate Reductase Probed by Asymmetric Mutations

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