Radical Redesign of a Tandem Array of Four R67 Dihydrofolate Reductase Genes Yields a Functional, Folded Protein Possessing 45 Substitutions

Biochemistry ◽  
2010 ◽  
Vol 49 (34) ◽  
pp. 7384-7392 ◽  
Author(s):  
Jian Feng ◽  
Jordan Grubbs ◽  
Ashita Dave ◽  
Sumit Goswami ◽  
Caroline Glyn Horner ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Tandem Array ◽  
R67 Dihydrofolate Reductase

Role of Ionic Interactions in Ligand Binding and Catalysis of R67 Dihydrofolate Reductase†

Biochemistry ◽  
2003 ◽  
Vol 42 (36) ◽  
pp. 10569-10578 ◽  
Author(s):  
Stephanie N. Hicks ◽  
R. Derike Smiley ◽  
J. Bradley Hamilton ◽  
Elizabeth E. Howell
Keyword(s):  
Ligand Binding ◽  
Dihydrofolate Reductase ◽  
Ionic Interactions ◽  
R67 Dihydrofolate Reductase

Does R67 Dihydrofolate Reductase Possess a Proton Donor?

1993 ◽  
pp. 493-498 ◽  
Author(s):  
Janel C. Holland ◽  
Charles E. Linn ◽  
Enrico DiGiammarino ◽  
Robert Nichols ◽  
Elizabeth E. Howell
Keyword(s):  
Dihydrofolate Reductase ◽  
Proton Donor ◽  
R67 Dihydrofolate Reductase

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

2004 ◽  
Vol 279 (45) ◽  
pp. 46995-47002 ◽  
Author(s):  
Stephanie N. Hicks ◽  
R. Derike Smiley ◽  
Lori G. Stinnett ◽  
Kenneth H. Minor ◽  
Elizabeth E. Howell
Keyword(s):  
Dihydrofolate Reductase ◽  
R67 Dihydrofolate Reductase

Further Studies on the Role of Water in R67 Dihydrofolate Reductase

Biochemistry ◽  
2013 ◽  
Vol 52 (12) ◽  
pp. 2118-2127 ◽  
Author(s):  
Mary Jane Timson ◽  
Michael R. Duff ◽  
Greyson Dickey ◽  
Arnold M. Saxton ◽  
José I. Reyes-De-Corcuera ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
R67 Dihydrofolate Reductase

Effects of Single-Tryptophan Mutations on R67 Dihydrofolate Reductase†

Biochemistry ◽  
2000 ◽  
Vol 39 (13) ◽  
pp. 3678-3689 ◽  
Author(s):  
Freddie W. West ◽  
Han-Seok Seo ◽  
Thomas D. Bradrick ◽  
Elizabeth E. Howell
Keyword(s):  
Dihydrofolate Reductase ◽  
R67 Dihydrofolate Reductase

scholarly journals Redesigning the Quaternary Structure of R67 Dihydrofolate Reductase

1996 ◽  
Vol 271 (45) ◽  
pp. 28031-28037 ◽  
Author(s):  
Thomas D. Bradrick ◽  
Chris Shattuck ◽  
Michael B. Strader ◽  
Carol Wicker ◽  
Edward Eisenstein ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Quaternary Structure ◽  
R67 Dihydrofolate Reductase

scholarly journals In VivoTitration of Folate Pathway Enzymes

2018 ◽  
Vol 84 (19) ◽  
Author(s):  
Deepika Nambiar ◽  
Timkhite-Kulu Berhane ◽  
Robert Shew ◽  
Bryan Schwarz ◽  
Michael R. Duff ◽  
...  
Keyword(s):  
Osmotic Stress ◽  
Dihydrofolate Reductase ◽  
Environmental Changes ◽  
Stress Conditions ◽  
Antibacterial Drug ◽  
Content Type ◽  
E Coli ◽  
Knockout Strain ◽  
R67 Dihydrofolate Reductase

ABSTRACTHow enzymes behave in cells is likely different from how they behave in the test tube. Previousin vitrostudies 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 occursin vivo, osmotic stress titrations were performed withEscherichia coli. 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 ΔfolAstrain, for a methylenetetrahydrofolate reductase clone rescuing a ΔmetFstrain, and for a serine hydroxymethyltransferase clone rescuing a ΔglyAstrain. Additionally, an R67 dihydrofolate reductase clone allowedE. coliDH5α 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.IMPORTANCEE. colican 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 inE. coliwas 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 thatE. colihas 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/cofactorsin vivo, and/or viscosity effects.

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

2004 ◽  
Vol 279 (45) ◽  
pp. 47003-47009 ◽  
Author(s):  
Lori G. Stinnett ◽  
R. Derike Smiley ◽  
Stephanie N. Hicks ◽  
Elizabeth E. Howell
Keyword(s):  
Ligand Binding ◽  
Dihydrofolate Reductase ◽  
R67 Dihydrofolate Reductase
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