Alternative mutations of a positively selected residue elicit gain or loss of functionalities in enzyme evolution

Abstract

All molecular species in an organism are connected physically and functionally to other molecules. In evolving systems, it is not obvious to what extent functional properties of a protein can change to selective advantage and leave intact favorable traits previously acquired. This uncertainty has particular significance in the evolution of novel pathways for detoxication, because an organism challenged with new xenobiotics in the environment may still require biotransformation of previously encountered toxins. Positive selection has been proposed as an evolutionary mechanism for facile adaptive responses of proteins to changing conditions. Here, we show, by saturation mutagenesis, that mutations of a hypervariable residue in human glutathione transferase M2-2 can differentially change the enzyme's substrate-activity profile with alternative substrates and, furthermore, enable or disable dissimilar chemical reactions. Crystal structures demonstrate that activity with epoxides is enabled through removal of steric hindrance from a methyl group, whereas activities with an orthoquinone and a nitroso donor are maintained in the variant enzymes. Given the diversity of cellular activities in which a single protein can be engaged, the selective transmutation of functional properties has general significance in molecular evolution.

Fig. 1.

Specific activities with six alternative substrates. (A) CDNB, cyanoDMNG, and aminochrome. (B) the epoxides tSBO, NPG, and SO. The different GST M2-2 variants have been divided into groups based on the character of the amino acid in position 210: aliphatic, hydroxyl-containing, sulfur-containing, aromatic, basic, and the acidic with their corresponding amides. Proline is separate because of its restricted side-chain mobility. All data are based on triplicate measurements. The standard deviations are <16% of the values, except for M2Thr and M2Val with NPG as substrate, where the standard deviations were 55% and 24%, respectively.

Fig. 2.

A three-dimensional substrate-activity space representing the activities of the GST M2-2 variants with CDNB, aminochrome, and tSBO. Mutations of residue 210 translocate the substrate-selectivity profile of the enzyme to different regions in multidimensional substrate-activity space. Starting from the wild-type enzyme M2Thr, the profile is differentially altered in M2Ser, M2Thr, M2Ala, M2Gly, and M2Pro; some alternative mutations (not indicated) reduce the activities with all three substrates.

Fig. 3.

Mechanism space spanned by different reaction categories: substitutions and additions. The substitution reactions can be further divided into aromatic substitution (CDNB) and transnitrosylation (cyanoDMNG). The addition reactions can be subdivided into Michael addition (aminochrome) and epoxide ring-opening (tSBO). Mutations of residue 210 disable or enable the four alternative chemical mechanisms in different combinations, depending on the amino acid replacement. The radar plots show specific activities (normalized to unit variance) with the alternative substrates.

Fig. 4.

Structure of the active site of GST M2-2 mutant M2Ser in complex with a glutathione conjugate of styrene-7,8-oxide. (A) The crystal structure of M2Ser determined at 1.35-Å resolution. Experimental 2Fo–Fc electron density map is shown for the ligand at 1σ contour level. Note the narrow channel below the phenyl ring of the ligand in M2Ser. (B) The modeled complex with the wild-type M2Thr structure (PDB ID code: 2GTU) showing steric interference between Thr in position 210 and the ligand.

Fig. 5.

Comparison of thermostabilities of the GST M2-2 variants. The orange bars represent half-lives at 48°C expressed in minutes. The insert shows four characteristic heat-inactivation curves. Half-lives differed as much as 500-fold among the mutants. M2Asn and M2Asp were the most thermostable variants, with half-lives of ≈1,000 min. The x axis indicates the alternative amino acids (one-letter codes) in position 210.

Fig. 6.

Distribution of amino acids occurring naturally at position 210 among the mammalian Mu-class GSTs. Polar residues are rendered as green bars, whereas hydrophobic residues are in red bars. The frequency of the codons for the different amino acids is also indicated.