Mechanism(s) of action of heavy metals to investigate the regulation of plastidic glucose-6-phosphate dehydrogenase

Alessia De Lillo,Simone Landi,Manuela Cardi,Sergio Esposito

doi:10.1038/s41598-018-31348-y

Alessia De Lillo, Simone Landi + Show 2 more

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https://doi.org/10.1038/s41598-018-31348-y

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Journal: Scientific Reports	Publication Date: Sep 7, 2018
Citations: 11	License type: open-access

Affiliation: University of Naples Federico II

Abstract

The regulation of recombinant plastidic glucose-6P dehydrogenase from Populus trichocarpa (PtP2-G6PDH - EC 1.1.1.49) was investigated by exposing wild type and mutagenized isoforms to heavy metals. Nickel and Cadmium caused a marked decrease in PtP2-G6PDH WT activity, suggesting their poisoning effect on plant enzymes; Lead (Pb++) was substantially ineffective. Copper (Cu++) and Zinc (Zn++) exposition resulted in strongest decrease in enzyme activity, thus suggesting a physiological competition with Magnesium, a well-known activator of G6PDH activity. Kinetic analyses confirmed a competitive inhibition by Copper, and a mixed inhibition by (Cd++). Mutagenized enzymes were differently affected by HMs: the reduction of disulfide (C175–C183) exposed the NADP+ binding sites to metals; C145 participates to NADP+ cofactor binding; C194 and C242 are proposed to play a role in the regulation of NADP+/NADPH binding. Copper (and possibly Zinc) is able to occupy competitively Magnesium (Mg++) sites and/or bind to NADP+, resulting in a reduced access of NADP+ sites on the enzyme. Hence, heavy metals could be used to describe specific roles of cysteine residues present in the primary protein sequence; these results are discussed to define the biochemical mechanism(s) of inhibition of plant plastidic G6PDH.

Highlights

In higher plants the oxidative pentose phosphate pathway (OPPP) plays multiple and fundamental roles, such as the synthesis of precursors for nucleic acids and fatty acids[1]; the furnishing of NADPH essential for primary metabolism[2,3], and to counteract oxidative stress[4,5,6]
WT and cysteine- mutagenized enzymes were overexpressed and successfully purified[21] (Supplementary Fig. S1), and kinetically characterized (Supplementary Table S1); the effects heavy metals (HMs) on PtP2-glucose-6-phosphate dehydrogenase (G6PDH) activity were investigated
In order to test the possible effect of his-tag on enzymatic activity, PtP2-G6PDH was alkylated, and this enzyme preparation showed over 92% of activity with respect to the purified his-tagged PtP2-G6PDH used throughout this work; we assumed that the histag did not affect the degree of inhibition by HMs

Summary

Introduction

In higher plants the oxidative pentose phosphate pathway (OPPP) plays multiple and fundamental roles, such as the synthesis of precursors for nucleic acids and fatty acids[1]; the furnishing of NADPH essential for primary metabolism[2,3], and to counteract oxidative stress[4,5,6]. The dependence on G6PDH reaction from Mg++ has been initially described in Bacteria[7,8]; it was suggested that Mg++ is able to affect the oligomeric state of the enzyme in dog liver G6PDH9 and human placental G6PDH10. In the root tissues, P2-G6PDH plays a pivotal role in providing reductants for anaplerotic metabolism, during nitrogen assimilation in non-photosynthetic plastids[2,16], and fatty acid synthesis[17]. G6PDHs from living organisms have been characterized from a number of sources, and kinetic properties of plant plastidic isoforms have been described previously[12,14,18,19,20,21]

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