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43012-AC4
Structure/Function Studies and Protein Engineering of ATP-Dependent Peptide Ligases
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 beta-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 beta-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 (Herrera et al., 2007).
Within the gamma-glutamylcysteine/glutathione-binding site, the S153A and S155A mutants displayed less than 4-fold changes in kinetic parameters with mutations of Glu220 (E220A/E220Q), Gln-226 (Q226A/Q226N), and Arg274 (R274A/R274K) at the distal end of the binding site resulting in 24-180-fold increases in the Km values for g-glutamylcysteine. Substitution of multiple residues interacting with ATP (K313M, K367M, and E429A/E429Q) or coordinating magnesium ions to ATP (E148A/E148Q, N150A/N150D, and E371A) yielded inactive protein because of compromised nucleotide binding, as determined by fluorescence titration. Other mutations in the ATP-binding site (E371Q, N376A, and K456M) resulted in greater than 30-fold decreases in affinity for ATP and up to 80-fold reductions in turnover rate. Mutation of Arg132 and Arg454, which are positioned at the interface of the two substrate-binding sites, affected the enzymatic activity differently. The R132A mutant was inactive, and the R132K mutant decreased kcat by 200-fold; however, both mutants bound ATP with Kd values similar to wild-type enzyme. Minimal changes in kinetic parameters were observed with the R454K mutant, but the R454A mutant displayed a 160-fold decrease in kcat. In addition, the R132K, R454A, and R454K mutations elevated the Km value for glycine up to 11-fold. Comparison of the pH profiles and the solvent deuterium isotope effects of AtGS and the Arg132 and Arg454 mutants also suggest distinct mechanistic roles for these residues. Based on these results, a catalytic mechanism for the eukaryotic GS is proposed.
During year 2, we have obtained 1.6 Å resolution x-ray diffraction data for GmhGS and a 1.5 Å resolution data set for the enzyme in complex with homo-glutathione and ADP. Modeling building and refinement of these structures are in progress.
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 (Hicks et al., 2007). 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.
