43012-AC4
Structure/Function Studies and Protein Engineering of ATP-Dependent Peptide Ligases
Year 3 (no cost extension) Summary
Cadmium, mercury, and lead pollute many industrial sites, such as refineries and natural gas plants, and are persistent bioaccumulative toxic compounds associated with a range of human health problems, including effects on the nervous system, reproductive and developmental problems, and carcinogenic effects. Understanding the enzymes involved in the biosynthesis of metal chelating peptides is essential for using plants as tools in the remediation of toxic metal contaminated soils and waters. In response to heavy metal toxicity, plants synthesize metal-chelating peptides (i.e., phytochelatins) derived from glutathione and related molecules as protection. Glutathione is found in mammals, plants, and bacteria and is synthesized by glutathione synthetase (GS), an ATP-dependent peptide ligase. Interestingly, some plants respond to heavy metal stress by synthesizing glutathione analogs in which β-alanine, serine, or glutamic acid replace glycine in the peptide. The specific aims of the proposal are as follows: (1) to determine the structural basis for the synthesis of glutathione analogs; (2) to probe the functional role of the substrate interaction loop; and (3) to diversify substrate specificity.
Aim 1: Structural studies. To elucidate the structural determinants of amino acid specificity, the three-dimensional structures of Arabidopsis thaliana GS (AtGS) and Glycine max (soybean) homo-GS (GmhGS), a β-alanine specific peptide ligase, will be compared. In year 1, we completed analysis of the 2.2 Å resolution crystal structure of AtGS in complex with ADP and glutathione. This structure set the stage for examining the reaction mechanism of the enzyme, during year 2 of the project (Herrera et al., 2007, J. Biol. Chem. 282: 17157-65).
Over the past year, we have determined the 1.9-2.1 Å resolution x-ray crystal structure of GmhGS in three forms: 1) apoenzyme/’open’ active site, 2) the ‘open’ form in complex with γ-glutamylcysteine, and 3) a ‘closed’ active site form in complex with homoglutathione and ADP. These structures shed light on domain movements occurring within GmhGS during its catalytic cycle. Comparison with GS suggests that two amino acid differences allow for accommodation of a larger substrate in hGS than GS. Site-directed mutagenesis of Leu466 and Pro467 within a conserved active site loop provides insight into the determinants of substrate specificity for β-alanine (hGS) versus glycine (GS) in these related enzymes. The structural studies of GmhGS and the protein engineering of substrate specificity described in Aim 2 are now being prepared for submission of a manuscript.
Aim 2: Glycine interaction loop in substrate specificity. Work on this aim was completed in year 1. Mutagenesis of AtGS to change Ala466 and Ala467 to the corresponding residues of GmhGS (A466L, A467P, and A466L/A467P) was performed. Likewise, amino acids in the interaction loop of GmhGS (Leu466 and Pro467) were mutated (L466A, P467A, L466A/P467A). In general, substitutions in either AtGS or GmhGS did not alter kinetic parameters for ATP or glutamylcysteine, but mutations of residues in both enzymes altered the catalytic efficiency (kcat/Km) for the third substrate and shifted the specificity of the enzyme.
Aim 3: Diversifying amino acid substrate specificity. In year 2, we initiated studies on another ATP-dependent peptide ligase (glutamate-cysteine ligase, GCL). Using a combination of mass spectrometry and site-directed mutagenesis, we examined the response of GCL to changes in redox environment. Mass spectrometry identified two disulfide bonds (Cys186-Cys406 and Cys349-Cys364) in GCL. Mutation of either Cys349 or Cys364 to a Ser reduced reaction rate by twofold, but substitution of a Ser for either Cys186 or Cys406 decreased activity by 20-fold and abrogated the response to changes in redox environment. Redox titrations show that the regulatory disulfide bond has a midpoint potential comparable with other known redox-responsive plant proteins. Modulation of activity depends on the Cys186-Cys406 disulfide bond. In vivo analysis of GCL in root extracts revealed that multiple oxidative stresses altered the distribution of oxidized (active) and reduced (inactive) enzyme and that this change correlated with increased GCL activity. The thiol-based regulation of GCL provides a posttranslational mechanism for modulating enzyme activity in response to in vivo redox environment and suggests a role for oxidative signaling in the maintenance of glutathione homeostasis in plants. These studies were published during year 3 (Hicks et al., 2007, Plant Cell 19: 2653-61).
