Elsevier

Algal Research

Volume 47, May 2020, 101890
Algal Research

Mapping the metal-catalytic site of a zinc-activated phytochelatin synthase

https://doi.org/10.1016/j.algal.2020.101890Get rights and content

Highlights

  • Metal catalytic site of Euglena phytochelatin synthase contains a C/H rich domain.

  • Catalysis and metallic substrate binding depend on a cysteine/histidine motif.

  • The Cys/His motif is not involved in binding of free glutathione.

Abstract

Phytochelatins are small enzyme-synthesized peptides that mediate tolerance to several heavy metals. To gain insight into the unusual metal catalytic site of the Euglena gracilis phytochelatin synthase (EgPCS), site-directed mutagenesis was performed on the C-terminal Cys/His (C/H) rich region 421-C-C-X-H-X-H-X-H-H-H-430. EgPCS mutants were analyzed at the cellular, kinetic and structural levels. Cd2+-resistance was conferred to Cd2+-sensitive yeast cells by EgPCS_wt (476aa) and truncated EgPCS_435 (435aa), but not by EgPCS_415 (415aa), suggesting that the resistance phenotype was linked to the enzyme C/H rich region. Moreover, full-length mutants (in which C and H residues in the C/H rich region were replaced by Ala) EgPCS_C2 (C421A/C422A) and EgPCS_C2H5 (C421A/C422A/H424A/H426A/H428A/H429A/H430A) did not provide Cd2+-resistance; in contrast, EgPCS_H2 (H424A/H426A), EgPCS_H3 (H428A/H429A/H430A), and EgPCS_H5 (H424A/H426A/H428A/H429A/H430A) mutants conferred similar Cd2+-tolerance like EgPCS_wt. Kinetic analysis showed that maximal rate (Vmax) for PC2 synthesis, affinity constants (Km Zn-GS2 or Km Cd-GS2) and catalytic efficiencies (Vmax/Km) were differentially impaired in the mutants, as compared to EgPCS_wt, with EgPCS_C2 being the most perturbed enzyme; however, the K0.5 values for GSH were not affected. All EgPCS mutants were predominantly monomeric. Far UV circular dichroism spectra and differential scanning calorimetry endotherms, indicated that alterations of the catalytic properties of EgPCS_C2 were not due to partial unfolding or destabilization of the native state of this mutant. The results indicated that C421/C422 and H424A/H426A/H428/H429/H430 are essential components of EgPCS for catalysis and activation by metal-substrate complexes.

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.

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