Evolutional Design of a Hyperactive Cysteine- and Methionine-free Mutant of Escherichia coli Dihydrofolate Reductase

Kosuke Maki,Kunihiko Gekko,Masahiro Iwakura,Tadashi Kamiyama,Hisashi Takahashi,Tatsuyuki Takenawa,Akiko Yokota,Katsuo Katayanagi

doi:10.1074/jbc.m508823200

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.

Highlights

To freely design enzymes with desired properties is the ultimate dream for protein engineers
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
When we set the goal of protein design “to create a protein of a desired property consisting of a given number of amino acid residues n,” the solution can be obtained by the complete search of all the possible amino acid sequences with “n” amino acid residues, the total number reaching 20n

Summary

Evolutional Design of DHFR

DHFR is a monomeric protein with two domains, which catalyzes the reduction of dihydrofolate to tetrahydrofolate by using the reducing cofactor NADPH [8]. In the course of such studies of DHFR, a number of variants of DHFR with a point mutation(s) at a site(s) within or in the vicinity of the active site were constructed [17,18,19,20,21,22,23]. Such mutational studies uncovered the roles of the residues around the active site. We describe the structural formation of ANLYF from its urea-induced unfolded state, suggesting that the folding kinetics would be conserved despite the significant change of the catalytic activity

EXPERIMENTAL PROCEDURES

RESULTS

Measured Calculated

Distances between folate and corresponding atoms of wild type or ANLYF

ANLYF koff

DISCUSSION