GLUTATHIONE TRANSFERASE (GST) SUPERFAMILY
Project period : 2010 - 2014
Richard Armstrong, Vanderbilt University School of Medicine
- generally nucleophilic attack of reduced glutathione (GSH) on an electrophlic substrate
- bi-domain structure containing a modified thioredoxin fold and an all α-helical domain
Challenges for Function Assignment
- promiscuous and catalytically diverse
- rarely found in operons eliminating functional clues from genomic context
- some members perform regulatory functions
- substrates may be small molecules or macromolecules (e.g. proteins)
Value to Integrated Strategy
- extreme sequence diversity drives advances in bioinformatic classification of distinct families
- pushes development of computational and bioinformatic strategies to predict functions in the absence of genomic context
- necessitates heavy use of in vitro tools to confirm physiological functions
- highly populated in Gram-negative, biologically tractable microorganisms facilitates in vivo analysis of function
GST superfamily members play key roles in prokaryotic and eukaryotic metabolism through utilization of reduced glutathione (GSH) to carry out a range of mechanistically diverse reactions. Traditionally classified into families based on a variety of criteria, cytosolic (soluble) GSTs are designated by Greek letters (e.g. alpha, mu, pi, sigma, theta, etc) with an additional class (kappa) describing mitochondrial GSTs. Recent bioinformatics analyses indicate approximately ~10,000 cytosolic GST superfamily members which can be split into two major sub-groups based on identifiable sequence/structural elements and active site architecture. These sub-groups are termed S/C-type and Y-type based on conservation of a key active site residue where the S/C-type sub-group encompasses the beta, omega, phi, tau, theta, and zeta classes while the Y-type sub-group includes the alpha, mu, pi, and sigma classes. Recently discovered enzymes in the Nu-class are Type-T enzymes that employ a threonine residue in this role.
Unlike many mechanistically diverse superfamilies, GSTs are unique in that sequence conservation appears to be driven by maintenance of fold stability instead of maintenance of catalytic features. As such, GST active sites are not strictly conserved. Instead, an extensive constellation of hydrogen bond partners throughout the first sphere and beyond may be responsible for divergent active sites being able to carry out similar reactions (Figure GST1).
Despite the low sequence identity (<10%) amongst GSTs, the tertiary and quaternary structures are remarkably consistent. All members of the GST superfamily contain an N-terminal thioredoxin-like fold and an α-helical C-terminal region (Figure GST2). Contacts for GSH binding are largely provided by the thioredoxin-like domain while the α-helical region generally defines the co-substrate binding pocket. Monomers oligomerize exclusively as homodimers to form the active enzyme although the nature of the interface (e.g. ball/socket, ionic, hydrophobic) varies between classes.
In accordance with the striking sequence diversity in the GST superfamily, the range of catalytic activities is correspondingly broad. GSTs have been show to carry out GSH-dependant nucleophilic substitution (SN2, SNAr), epoxide ring opening, conjugate addition, ester thiolysis, isomerization, disulfide-bond reduction, nitrate ester reduction, hydroperoxidase reactions (e.g. Figure GST3). Although the vast majority of GST superfamily members require GSH as a co-substrate, a few discoveries of GSH-independent reactions have been observed, such as the isomerization of 13-cis-retinoic acid to all-trans retinoic acid by an anomalous Pi class GST from humans. Non-catalytic functions including intracellular transport and transcriptional regulation have also been described further accentuating the extreme diversity within the GST superfamily.
While substrate/chemistry-specific residues vary by class and family, requisite to GSH-dependent catalysis is activation of the thiol for subsequent nucleophilic attack. The active site residues primarily responsible for GSH activation are localized at end of the first β strand (Tyr) or in the following loop (Ser/Cys), which gives rise to the GST sub-group nomenclature mentioned above. Based on the low fraction of ionized Tyr at neutral pH coupled with deuterium isotope effect experiments and incorporation of fluoro-Tyr residues, general base catalysis has been ruled out for the role of this position. Instead it is believed that the side chain groups act as hydrogen bond donors to activate GSH. This is supported by the observation that the pKa of GSH is substantially lower in the active site (6.0-7.5) than in solution (9.0) indicating the thiolate persists as the predominant species.
Initially recognized for the ability to detoxify xenobiotics, members of the GST superfamily are now recognized to play significant roles in an array of cellular metabolism. Broad substrate specificity/reactivity coupled with roles in general responses to cellular stress muddles the assignment of physiological functions for dedicated GSTs. For example, only 1 of the 9 E. coli GSTs have an established function. Although daunting, these hurdles will push the Superfamily/Genome and Computational Cores to develop more sophisticated methods for in silico functional predictions as well as faciliate development of the Microbiology Core for confirmation of in vitro activities. For these reasons the GST superfamily offers a highly instructive system for functional assignment within the EFI.
- Structure, catalytic mechanism, and evolution of the glutathione transferases. Armstrong RN. (1997) Chem Res Toxicol 10, 2-18.
- Recruitment of a double bond isomerase to serve as a reductive dehalogenase during biodegradation of pentachlorophenol. Anandarajah K, Kiefer PM Jr, Donohoe BS, Copley SD. (2000) Biochemistry 39, 5303-11.
- Structure, function and evolution of glutathione transferases: implications for classification of non-mammalian members of an ancient enzyme superfamily. Sheehan D, Meade G, Foley VM, Dowd CA. (2001) Biochem J 360, 1-16.
- Glutathione transferases are structural and functional outliers in the thioredoxin fold. Atkinson HJ, Babbitt PC. (2009) Biochemistry 48, 11108-16.