“Catch 222,” the Effects of Symmetry on Ligand Binding and Catalysis in R67 Dihydrofolate Reductase as Determined by Mutations at Tyr-69

Stinnett, Lori G.; Smiley, R. Derike; Hicks, Stephanie; Howell, Elizabeth E.

doi:10.1074/jbc.m404485200

Cited by 12 publications

(13 citation statements)

References 38 publications

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“…The first asymmetric mutation studied was Q67H (6). This study focuses on the role of Lys-32 residues, and a third study considers the effect of Y69F mutations (see companion paper: Stinnett et al (46)). …”

Section: R67 Dihydrofolate Reductase (R67 Dhfr)mentioning

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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Section: R67 Dihydrofolate Reductase (R67 Dhfr)mentioning

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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“…However, using osmotic pressure, we could titrate the ability of DH5␣ carrying a Y69L R67 DHFR clone to confer resistance to TMP. The Y69L mutant enzyme has levels of activity intermediate between those of the R67 DHFR and the K32M mutant (9,33). This pattern of behavior indicates a window of enzyme activity that allows cell growth and selection by osmotic stress.…”

Section: Resultsmentioning

confidence: 95%

In Vivo Titration of Folate Pathway Enzymes

Nambiar

Berhane

Shew

et al. 2018

Appl Environ Microbiol

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How enzymes behave in cells is likely different from how they behave in the test tube. Previous studies find that osmolytes interact weakly with folate. Removal of the osmolyte from the solvation shell of folate is more difficult than removal of water, which weakens binding of folate to its enzyme partners. To examine if this phenomenon occurs, osmotic stress titrations were performed with Two strategies were employed: resistance to an antibacterial drug and complementation of a knockout strain by the appropriate gene cloned into a plasmid that allows tight control of expression levels as well as labeling by a degradation tag. The abilities of the knockout and complemented strains to grow under osmotic stress were compared. Typically, the knockout strain could grow to high osmolalities on supplemented medium, while the complemented strain stopped growing at lower osmolalities on minimal medium. This pattern was observed for an R67 dihydrofolate reductase clone rescuing a Δ strain, for a methylenetetrahydrofolate reductase clone rescuing a Δ strain, and for a serine hydroxymethyltransferase clone rescuing a Δ strain. Additionally, an R67 dihydrofolate reductase clone allowed DH5α to grow in the presence of trimethoprim until an osmolality of ∼0.81 is reached, while cells in a control titration lacking antibiotic could grow to 1.90 osmol. can survive in drought and flooding conditions and can tolerate large changes in osmolality. However, the cell processes that limit bacterial growth under high osmotic stress conditions are not known. In this study, the dose of four different enzymes in was decreased by using deletion strains complemented by the gene carried in a tunable plasmid. Under conditions of limiting enzyme concentration (lower than that achieved by chromosomal gene expression), cell growth can be blocked by osmotic stress conditions that are normally tolerated. These observations indicate that has evolved to deal with variations in its osmotic environment and that normal protein levels are sufficient to buffer the cell from environmental changes. Additional factors involved in the osmotic pressure response may include altered protein concentration/activity levels, weak solute interactions with ligands which can make it more difficult for proteins to bind their substrates/inhibitors/cofactors , and/or viscosity effects.

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“…The mobility of the tail regions has been observed not only in both X-ray crystallography and NMR studies but is also in agreement with mutational analysis. Asymmetric mutations of Y69 indicate that variable positions of the pABA-Glu tail of DHF are tolerated [26], and studies of K32 showed that two mutations on different half pores produce topologies that allow comparable interactions between K32 and the Glu tail of DHF [20]. Furthermore, mutations at the centre of the pore (Q67H) are accompanied by inhibition of the catalytic activity due to non-productive binding [70], while mutations close to the pore surface (K32M and Y69F) are not.…”

Section: Multiple Conformationsmentioning

confidence: 99%

“…molecular dynamics, MD). Many new experimental studies have been conducted on R67 DHFR since the publication of this initial study [17,19,20,[25][26][27], providing new insights into the mechanism R67 DHFR uses to catalyse the reaction. Some of this new data, such as the interaction between the hydroxyl group of Y69 and NADPH observed in mutational analysis [25], is not in agreement with the previous docking results [21] and, therefore, this model requires re-examination.…”

Section: Introductionmentioning

confidence: 98%

Multiple ligand-binding modes in bacterial R67 dihydrofolate reductase

Alonso

Gillies

Cummins

et al. 2005

J Comput Aided Mol Des

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R67 dihydrofolate reductase (DHFR), a bacterial plasmid-encoded enzyme associated with resistance to the drug trimethoprim, shows neither sequence nor structural homology with the chromosomal DHFR. It presents a highly symmetrical toroidal structure, where four identical monomers contribute to the unique central active-site pore. Two reactants (dihydrofolate, DHF), two cofactors (NADPH) or one of each (R67*DHF*NADPH) can be found simultaneously within the active site, the last one being the reactive ternary complex. As the positioning of the ligands has proven elusive to empirical determination, we addressed the problem from a theoretical perspective. Several potential structures of the ternary complex were generated using the docking programs AutoDock and FlexX. The variability among the final poses, many of which conformed to experimental data, prompted us to perform a comparative scoring analysis and molecular dynamics simulations to assess the stability of the complexes. Analysis of ligand-ligand and ligand-protein interactions along the 4 ns trajectories of eight different structures allowed us to identify important inter-ligand contacts and key protein residues. Our results, combined with published empirical data, clearly suggest that multipe binding modes of the ligands are possible within R67 DHFR. While the pterin ring of DHF and the nicotinamide ring of NADPH assume a stacked endo-conformation at the centre of the pore, probably assisted by V66, Q67 and I68, the tails of the molecules extend towards opposite ends of the cavity, adopting multiple configurations in a solvent rich-environment where hydrogen-bond interactions with K32 and Y69 may play important roles.

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“Catch 222,” the Effects of Symmetry on Ligand Binding and Catalysis in R67 Dihydrofolate Reductase as Determined by Mutations at Tyr-69

Cited by 12 publications

References 38 publications

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

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

In Vivo Titration of Folate Pathway Enzymes

Multiple ligand-binding modes in bacterial R67 dihydrofolate reductase

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