Mapping the metal-catalytic site of a zinc-activated phytochelatin synthase
Graphical abstract
Introduction
Phytochelatin synthase (PCS) provides resistance against heavy metal toxicity by synthesizing metal-chelating polymers called phytochelatins (PCs), commonly from PC2 ([Glu-Cys]2-Gly) to PC6 ([Glu-Cys]6-Gly) units. PCS uses GSH and different metal-bis-glutathionate (Me-GS2) complexes as substrates to produce PCs, which in turn bind and inactivate metals intracellularly; the metal-PCs complexes may be further compartmentalized into vacuoles, chloroplasts and mitochondria [1]. PCs synthesis is mainly induced in plants and other organisms by cadmium, although PC synthesis can also be triggered to lower extents by arsenite, mercury, lead, zinc, copper, nickel, silver and chromium [[2], [3], [4], [5], [6], [7], [8]]. The molecular and kinetic mechanisms that determine the metal-dependence of phytochelatin synthases (PCSs) have not been yet elucidated. Understanding these mechanisms is important to engineer PCSs efficiency to thus improving their performance and consequently the accumulation/traffic/storage processes of essential- and non-essential metals in plants and microorganisms [1,9,10].
The Arabidopsis thaliana PCS1 (AtPCS1) is perhaps one of the best characterized PC synthases. For instance, the activity (Vmax) of AtPCS1 in presence of GSH and cadmium-bis-glutathionate (Cd-GS2) is higher than with zinc-bis-glutathionate (Zn-GS2) as co-substrate, despite displaying lower affinity for Cd-GS2 (Km = 9.2 μM) than for Zn-GS2 (Km = 4.5 μM) [11]. Analyses of truncated versions of AtPCS also suggests that the metal-catalytic site (MCS) responsible for cadmium activation resides in the 221 aa segment of the amino-terminal domain (N-ter) [12,13], while the carboxyl-terminal domain (C-ter) segments Ser373 to His460 and Leu459 to Arg470 are involved in the MCS activated by zinc [14] and arsenic-III [10], respectively. As the AtPCS truncated versions were not kinetically characterized and the AtPCS crystal structure has not been resolved, it remains elusive whether the proposed MCS residues contribute to (i) recognition of the metallic-substrate, (ii) catalysis, and/or (iii) a metal activation site. No other PCS from plants or other organisms have been molecularly analyzed at this level.
PCS from the green alga-like Euglena gracilis (EgPCS) achieves maximal activity in presence of GSH and Zn-GS2 rather than Cd-GS2 [15]. EgPCS has a Cys/His (C/H) rich region (Cys421, Cys422, His424, His426, His428, His429 and His430) in the C-ter (Fig. 1), which is similar to the C/H rich regions of zinc-proteins such as Cu/Zn superoxide dismutase from Neisseria meningitidis (His104, His113, His122 and Asp125) [16]. His-rich regions also play critical roles in the transport of heavy metal ions, such as the His-rich extra membrane loop of Zn-transporter ZIP1 from Glycine max [17], as well as in Zinc-finger proteins [18] and certain hydrolases [19]. Therefore, the C/H rich region of EgPCS (Fig. 1) may also be of critical importance for metallic-substrate binding (e.g. Zn-GS2, Cd-GS2). Thus, the aim of the present study was to identify essential residues of MCS in EgPCS. To this end, the molecular and kinetic characterization of several variants of EgPCS carrying either deletions or substitutions at each of the Cys and His residues within the EgPCS C-terminus was undertaken.
Section snippets
Chemicals
Yeast nitrogen base medium (BD, Sparks Glencoe, MD, USA), yeast synthetic drop-out medium without uracil (Sigma Aldrich; St. Louis, MO, USA), and solutions of galactose (Gal, Sigma Aldrich) plus raffinose (Raff, Sigma Aldrich) and CdCl2 were sterilized by filtration through 0.22 μm pore-diameter membrane filters (Millipore). Analytical grade stock solutions of ZnCl2, CdCl2, NiCl2, CuSO4, FeCl2, MnCl2, AgNO3 and NaAsO3 were calibrated by atomic absorption spectrophotometry (Varian SpectraAA-640,
EgPCS amino acid sequence analysis
An update of the alignment previously shown in [15] was performed. The results showed coincidences with sequences that have already been reported as PCSs, as well as with other putative proteins annotated with this function and hypothetical proteins. The complete EgPCS sequence showed the highest similarities with annotated PCSs from cyanobacteria and some fungi (Fig. S1), although these proteins have not been functionally characterized as PCSs. The alignment confirmed that the EgPCS C-ter is a
Discussion
PCs biosynthesis catalyzed by PCSs is intimately linked to heavy metal resistance mechanisms evolved in some microorganisms and plants. For PCS to become active, the presence of a heavy metal ion such as Cd2+, Zn2+, Cu2+, Hg2+ and Pb2+ is compulsory.
Analysis of the MCS of AtPCS has suggested that the sensing regions for Zn2+, Cd2+ and As3+ are localized in the C-ter domain [10,14]. However, as such regions are not essential for AtPCS activity [7,10,14], it has been proposed that MCS of AtPCS
Statement of informed consent, human/animal rights
No conflicts, informed consent, or human or animal rights are applicable to this study.
CRediT authorship contribution statement
J.D. García-García: Conceptualization, Visualization, Methodology, Investigation, Formal analysis, Writing - original draft. R. Sánchez-Thomas: Investigation, Visualization, Formal analysis, Validation, Writing - review & editing. E. Saavedra: Visualization, Resources, Supervision, Writing - review & editing. D.A. Fernández-Velasco: Methodology, Resources, Writing - review & editing. S. Romero-Romero: Methodology, Investigation. K.I. Casanova-Figueroa: Methodology, Investigation. D.G.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgements
The present work was partially supported by grants Nos., 239930, 281428 and 282663 from CONACYT-México. The authors acknowledge Dr. Miguel Costas (Facultad de Química, UNAM) for the use of the DSC capillary-calorimeter.
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Current address: Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611, USA.