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Mutagenic Definition of a Papain-Like Catalytic Triad, Sufficiency of the N-Terminal Domain for Single-Site Core Catalytic Enzyme Acylation, and C-Terminal Domain for Augmentative Metal Activation of a Eukaryotic Phytochelatin Synthase
- Source :
- Plant Physiology. 141:858-869
- Publication Year :
- 2006
- Publisher :
- Oxford University Press (OUP), 2006.
-
Abstract
- Phytochelatin (PC) synthases are γ-glutamylcysteine (γ-Glu-Cys) dipeptidyl transpeptidases that catalyze the synthesis of heavy metal-binding PCs, (γ-Glu-Cys)nGly polymers, from glutathione (GSH) and/or shorter chain PCs. Here it is shown through investigations of the enzyme from Arabidopsis (Arabidopsis thaliana; AtPCS1) that, although the N-terminal half of the protein, alone, is sufficient for core catalysis through the formation of a single-site enzyme acyl intermediate, it is not sufficient for acylation at a second site and augmentative stimulation by free Cd2+. A purified N-terminally hexahistidinyl-tagged AtPCS1 truncate containing only the first 221 N-terminal amino acid residues of the enzyme (HIS-AtPCS1_221tr) is competent in the synthesis of PCs from GSH in media containing Cd2+ or the synthesis of S-methyl-PCs from S-methylglutathione in media devoid of heavy metal ions. However, whereas its full-length hexahistidinyl-tagged equivalent, HIS-AtPCS1, undergoes γ-Glu-Cys acylation at two sites during the Cd2+-dependent synthesis of PCs from GSH and is stimulated by free Cd2+ when synthesizing S-methyl-PCs from S-methylglutathione, HIS-AtPCS1_221tr undergoes γ-Glu-Cys acylation at only one site when GSH is the substrate and is not directly stimulated, but instead inhibited, by free Cd2+ when S-methylglutathione is the substrate. Through the application of sequence search algorithms capable of detecting distant homologies, work we reported briefly before but not in its entirety, it has been determined that the N-terminal half of AtPCS1 and its equivalents from other sources have the hallmarks of a papain-like, Clan CA Cys protease. Whereas the fold assignment deduced from these analyses, which substantiates and is substantiated by the recent determination of the crystal structure of a distant prokaryotic PC synthase homolog from the cyanobacterium Nostoc, is capable of explaining the strict requirement for a conserved Cys residue, Cys-56 in the case of AtPCS1, for formation of the biosynthetically competent γ-Glu-Cys enzyme acyl intermediate, the primary data from experiments directed at determining whether the other two residues, His-162 and Asp-180 of the putative papain-like catalytic triad of AtPCS1, are essential for catalysis have yet to be presented. This shortfall in our basic understanding of AtPCS1 is addressed here by the results of systematic site-directed mutagenesis studies that demonstrate that not only Cys-56 but also His-162 and Asp-180 are indeed required for net PC synthesis. It is therefore established experimentally that AtPCS1 and, by implication, other eukaryotic PC synthases are papain Cys protease superfamily members but ones, unlike their prokaryotic counterparts, which, in addition to having a papain-like N-terminal catalytic domain that undergoes primary γ-Glu-Cys acylation, contain an auxiliary metal-sensing C-terminal domain that undergoes secondary γ-Glu-Cys acylation.
- Subjects :
- Physiology
Stereochemistry
Acylation
medicine.medical_treatment
Molecular Sequence Data
Arabidopsis
Plant Science
Catalysis
chemistry.chemical_compound
Papain
Catalytic triad
Genetics
medicine
Histidine
Amino Acid Sequence
Cysteine
chemistry.chemical_classification
Aspartic Acid
Binding Sites
Protease
Sequence Homology, Amino Acid
ATP synthase
biology
Chemistry
C-terminus
Aminoacyltransferases
Protein Structure, Tertiary
Enzyme Activation
Enzyme
Biochemistry
Mutagenesis, Site-Directed
biology.protein
Phytochelatin
Cadmium
Research Article
Subjects
Details
- ISSN :
- 15322548 and 00320889
- Volume :
- 141
- Database :
- OpenAIRE
- Journal :
- Plant Physiology
- Accession number :
- edsair.doi.dedup.....490aa545c5c353aba4843e5b597c1f80
- Full Text :
- https://doi.org/10.1104/pp.106.082131