2d0k | pdb_00002d0k

Methionine-free mutant of Escherichia coli dihydrofolate reductase

spinqualitylabelsall models 2d0k, resolution 1.90Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 2d0k is a 2 chain structure with sequence from Escherichia coli. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Method: X-ray diffraction, Resolution 1.9Å Ligands: CL, FOL Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Function DYR_ECOLI Key enzyme in folate metabolism. Catalyzes an essential reaction for de novo glycine and purine synthesis, and for DNA precursor synthesis. Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMed We developed a strategy for finding out the adapted variants of enzymes, and we applied it to an enzyme, dihydrofolate reductase (DHFR), in terms of its catalytic activity so that we successfully obtained several hyperactive cysteine- and methionine-free variants of DHFR in which all five methionyl and two cysteinyl residues were replaced by other amino acid residues. Among them, a variant (M1A/M16N/M20L/M42Y/C85A/M92F/C152S), named as ANLYF, has an approximately seven times higher k(cat) value than wild type DHFR. Enzyme kinetics and crystal structures of the variant were investigated for elucidating the mechanism of the hyperactivity. Steady-state and transient binding kinetics of the variant indicated that the kinetic scheme of the catalytic cycle of ANLYF was essentially the same as that of wild type, showing that the hyperactivity was brought about by an increase of the dissociation rate constants of tetrahydrofolate from the enzyme-NADPH-tetrahydrofolate ternary complex. The crystal structure of the variant, solved and refined to an R factor of 0.205 at 1.9-angstroms resolution, indicated that an increased structural flexibility of the variant and an increased size of the N-(p-aminobenzoyl)-L-glutamate binding cleft induced the increase of the dissociation constant. This was consistent with a large compressibility (volume fluctuation) of the variant. A comparison of folding kinetics between wild type and the variant showed that the folding of these two enzymes was similar to each other, suggesting that the activity enhancement of the enzyme can be attained without drastic changes of the folding mechanism. Evolutional design of a hyperactive cysteine- and methionine-free mutant of Escherichia coli dihydrofolate reductase.,Iwakura M, Maki K, Takahashi H, Takenawa T, Yokota A, Katayanagi K, Kamiyama T, Gekko K J Biol Chem. 2006 May 12;281(19):13234-46. Epub 2006 Mar 1. PMID:16510443 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See Also Dihydrofolate reductase 3D structures

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