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-Å 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.To freely design enzymes with desired properties is the ultimate dream for protein engineers. Because the "protein folding problem," namely how an amino acid sequence determines its tertiary folded structure, has not completely been solved at atomic resolution, the rational design approach is still limited, even for improvement of enzymes from natural sources. Alternatively, protein design can be thought of as a process of "picking up" from all the possible amino acid sequences (i.e. sequence space) until a protein with the desired properties is found, because the structure and function of a protein is determined by its amino acid sequence (1). 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 20 n . Therefore, the protein design problem can be reduced to a searching problem in sequence space of polypeptides with n amino acid residues. The solution to this searching problem should include a reliable process that can be performed within a realistic time span, otherwise it is of no use in practical protein design. In this regard, the size of sequence space to be searched is a critical factor (2). The size of the sequence space of a polypeptide with amino acids even as small as 100 is ...

A Systematic and Comprehensive Combinatorial Approach to Simultaneously Improve the Activity, Reaction Specificity, and Thermal Stability of p-Hydroxybenzoate Hydroxylase

“…Indeed, it was recently reported that many protein functions are not confined to a small set of amino acids but rather can be affected by residues far away from the enzyme active site (54). These results suggest that the QAW method must be useful for searching for mutants with more improved functions with poor structural information on the target enzyme (1).…”

“…As recently reported, our evolutional design method, namely, "quasi-additive adaptive walking with mutant data base" (QAW), 2 provides a systematic and comprehensive combinatorial scheme for obtaining an improved protein with a single desired property (1). Because the previous work focused on a single property (enzyme activity), whether or not our QAW method could be extended to improve simultaneously multiple properties of any enzyme should be examined for generalization and availability as to industrial uses of our method.…”

“…Because the previous work focused on a single property (enzyme activity), whether or not our QAW method could be extended to improve simultaneously multiple properties of any enzyme should be examined for generalization and availability as to industrial uses of our method. In a test study, we used p-hydroxybenzoate hydroxylase (PHBH) as a target enzyme and all Cys and Met residues as mutation sites, similar to the previous study (1).…”

“…Finally, among the 12 Cys and Met sites, the replacement positions numbered 8 (Cys-152, Cys-211, Cys-332, Met-52, Met-110, Met-213, Met-276, and Met-349) and seven (Cys-152, Cys-211, Cys-332, Met-52, Met-110, Met-213, and Met-276), respectively, in the selected single mutants and the obtained quadruple variants through combination using four selection parameters. Regarding useful systematic improvement of a target enzyme, the combinatorial method makes it possible to carry out effective adaptive walking while reducing the sequence space that must be searched (1).…”

“…When we attempted to obtain a Cys-and Met-reduced PHBH with increased main activity, reduced sub-activity, reduced reaction specificity, and increased thermal stability as compared with those of the wild type by creating mutant libraries by random mutagenesis, before starting the experiments described in this paper, such variants could not been obtained. As mentioned above, the QAW strategy should be significantly potent and effective in cases where it is inefficient to construct mutant pools and to screen them by means of directed evolution strategies (1).…”