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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

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 Synthase¹

Plant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104 (N.D.R., O.K.V., A.L., P.A.R.); School of Biological Sciences, University of Liverpool, Liverpool L69 7ZB, United Kingdom (D.J.R.); and Donald Danforth Plant Science Center, St. Louis, Missouri 63132 (R.E.C., J.M.J.)

Phytochelatin (PC) synthases are -glutamylcysteine (-Glu-Cys) dipeptidyl transpeptidases that catalyze the synthesis of heavy metal-binding PCs, (-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 -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 -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 -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 -Glu-Cys acylation, contain an auxiliary metal-sensing C-terminal domain that undergoes secondary -Glu-Cys acylation.

² These authors contributed equally to the paper.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Philip A. Rea (parea{at}sas.upenn.edu).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.082131.

^* Corresponding author; e-mail parea{at}sas.upenn.edu; fax 215–898–8780.

Received April 15, 2006; returned for revision May 9, 2006; accepted May 9, 2006.

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