title: Evolutional design of a hyperactive cysteine- and methionine-free mutant of Escherichia coli dihydrofolate reductase.
abstract: 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.
authors.affiliation: National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan. Electronic address: masa-iwakura@aist.go.jp.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Iwakura
authors.affiliation: National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Kosuke
authors.initials: K
authors.lastname: Maki
authors.affiliation: National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Takahashi
authors.affiliation: National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Tatsuyuki
authors.initials: T
authors.lastname: Takenawa
authors.affiliation: National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Yokota
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan.
authors.forename: Katsuo
authors.initials: K
authors.lastname: Katayanagi
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Kamiyama
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan.
authors.forename: Kunihiko
authors.initials: K
authors.lastname: Gekko
mesh_terms: D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry*; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008715:Methionine / Q000737:chemistry*; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D008715:Methionine; D013762:Tetrahydrofolate Dehydrogenase; D003545:Cysteine