Minor Modifications of the C-terminal Helix Reschedule the Favored Chemical Reactions Catalyzed by Theta Class Glutathione Transferase T1-1

Abstract

Adaptive responses to novel toxic challenges provide selective advantages to organisms in evolution. Glutathione transferases (GSTs) play a pivotal role in the cellular defense because they are main contributors to the inactivation of genotoxic compounds of exogenous as well as of endogenous origins. GSTs are promiscuous enzymes catalyzing a variety of chemical reactions with numerous alternative substrates. Despite broad substrate acceptance, individual GSTs display pronounced selectivities such that only a limited number of substrates are transformed with high catalytic efficiency. The present study shows that minor structural changes in the C-terminal helix of mouse GST T1-1 induce major changes in the substrate-activity profile of the enzyme to favor novel chemical reactions and to suppress other reactions catalyzed by the parental enzyme.The molecular evolution of enzyme functions encompasses redesign of existing structures as one of the cornerstones (1). Structural studies and evolutionary analyses give ample examples of structural scaffolds that have been adopted for diverse functions (2). Many enzymes have evolved to acquire high selectivity for naturally occurring substrates, but enzymes involved in the cellular detoxication system generally have a broad substrate acceptance, or promiscuity (3). Proteins have high structural flexibility, and in some enzymes the active site can undergo major conformational changes and thereby accommodate substrates of widely different shapes, as exemplified by cytochrome P450 3A4 (4). Glutathione transferases (GSTs) 2 form a superfamily of enzymes encompassing seven classes of canonical soluble enzymes (5). Some of the GSTs have evolved for intermediary metabolism, such as the biosynthesis of steroid hormones (6) and prostaglandins (7), or the degradation of aromatic acids (8). However, the majority of the soluble GSTs appear to have their primary function in the inactivation of toxic compounds from exogenous as well as endogenous sources.Surprisingly, structurally highly similar GST homologs in the same biological organism or from diverse species differ in their substrate selectivity profiles. A primary determinant for divergent substrate acceptance among GSTs appears to be the H-site, i.e. the binding site for the electrophilic substrate (9, 10). On the other hand, GST A4-4 in rats and mice are structurally quite different from the human counterpart, even though their catalytic efficiencies are similarly evolved for high activity with the cytotoxic 4-hydroxynonenal generated by lipid peroxidation (11). These findings suggest that the GST structure is functionally malleable such that catalytic properties can readily be altered in a relatively short evolutionary time span. In the present investigation we demonstrate how simple point mutations and a deletion in the C-terminal helix of Theta class GST (GST T1-1) can drastically alter the substrate specificity profile with consequent increases in activity with certain substrates and decreases with other substrates. This shi...

Effects of Substrate-Binding Site Residues on the Biochemical Properties of a Tau Class Glutathione S-Transferase from Oryza sativa

“…In view of the large space occupied by cumene hydroperoxide, we speculated that the residues of the H-site hindered the entry of cumene hydroperoxide into the H-site. Strikingly, a replacement of Met232, which the side chain pointing away from the pocket, by Ala in mice GSTT1-1 led to an approximately 30-fold increase in catalytic activity toward cumene hydroperoxide [44]. This result implies that residues affecting the substrate activity of cumene hydroperoxide are not necessarily those composing the H-site.…”

Metabolism of isothiocyanates in individuals with positive and null GSTT1 and M1 genotypes after drinking watercress juice

“…First, GSTT1 and M1 do not play a significant role as a rate determinant step in ITC conjugation; second, PEITC, a major component in watercress, is not a good substrate for GSTT1 and M1 ; and third, genes other than GST in the mercapturic acid pathway may be more important in metabolizing ITCs (35, 36). The question is why individuals with GSTT1 and M1 null genotypes are better protected.…”

Exploring sequence-function space of a poplar glutathione transferase using designed information-rich gene variants

“…This outcome could be explained by the fact that the infologs in our design did not target the active-site residues, but other residues known from homologous GST proteins to be mutable. Substantial changes in substrate selectivities obtained by mutations of substrate-contacting amino acids ( Nilsson et al , 2000 ; Pettersson et al , 2002 ; Ivarsson et al , 2003 ; Norrgård et al , 2006 ; Blikstad et al , 2008 ; Shokeer and Mannervik, 2010 ) suggest the prospect of altering the substrate selectivity also of GSTU45. Incisive application of the infolog approach to the H-site residues may be similarly effective for this purpose as in boosting existing activities as shown in the present investigation.…”