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 substrateactivity 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.enabling alternative reactions ͉ glutathione transferase ͉ GST ͉ positive selection ͉ saturation mutagenesis T he evolution of protein functions is a multiparameter optimization that is constrained by boundary conditions set by ambient physical conditions, interactions with other molecules, and possible alternative functions. Expressivity of the cognate gene in the ribosomal translation system and solubility and stability of the protein in the cellular milieu are obvious restrictions. Furthermore, it is estimated that Ͼ50% of the human genes undergo alternative splicing (1), and the change in the coding sequence for a protein may jeopardize the function of an alternatively spliced mRNA. These and other limitations attenuate mutational changes that otherwise would be feasible and, presumably, partly explain why molecular structures in biological systems are highly conserved. This study shows how point mutations in a single positively selected position can lead to drastic changes of both substrate selectivities and physical properties of an enzyme and indicates how a protein could rapidly respond in multiple dimensions of functional space, optionally with minimal effects on alternative functions of the protein.GSTs are members of a diverse superfamily of detoxication enzymes. The GSTs have evolved through divergent evolution from a common ancestor, and the enzymes are divided into classes based on similarities in the primary structure (2-4). As detoxication enzymes, GSTs are endowed with broad substrate specificities and functional plasticity. These qualities are valuable in providing the organism with protection against various harmful chemical species but may also be important parameters contribu...

Structural and Functional Comparisons of Nucleotide Pyrophosphatase/Phosphodiesterase and Alkaline Phosphatase: Implications for Mechanism and Evolution,

“…There is a growing number of examples of promiscuous activities that can be substantially enhanced with one mutation or a few mutations (72)(73)(74)(75)(76)(77)(78). In some cases, these enhancements occur without significant detrimental effects on the native function, suggesting that optimization of a new enzyme activity prior to gene duplication could sometimes occur without negative effects on organism fitness (74).…”

Engineering GST M2-2 for High Activity with Indene 1,2-Oxide and Indication of an H-Site Residue Sustaining Catalytic Promiscuity

“…3 In human GST M2-2, this residue is Thr210, and mutations of Thr into other residues change the activities with alternative substrates in a differential manner. 4 The most remarkable alterations were the conspicuously enhanced activities of GST M2-2 with epoxides that were afforded by the substitution of Thr with the smaller amino acid residue Gly, Ala, or Ser.…”

Cys-X Scanning for Expansion of Active-site Residues and Modulation of Catalytic Functions in a Glutathione Transferase

“…The purified protein was dialyzed against the equilibration buffer containing 1 mM 2-mercaptoethanol. Thermal inactivation studies were made in PBS for 1 h to assess the stability of the C87A/C115A/C174A/M212C mutant at 48°C and the remaining activity was assayed with 1-chloro-2,4-dinitrobenzene (CDNB) (7).…”

“…2 GSTs catalyze the conjugation of glutathione with a broad range of alternative electrophiles and are structurally and functionally well characterized (5,6). hGST M2-2 is well suited as an example of the potential of Cys-X scanning because it has widely different catalytic efficiencies with alternative electrophilic substrates, which are undergoing divergent types of chemical reactions (7,8). We demonstrate that different substitutions of Cys in the substrate-binding site in some cases actually enhance the catalytic activity with a particular substrate.…”