Cloning and characterisation of S-formylglutathione hydrolase from Arabidopsis thaliana: a pathway for formaldehyde detoxification

Abstract

The toxic accumulation of formaldehyde in plant cells can occur from a number of sources: atmospheric pollution, endogenous P450 enzymes de-methylating herbicides and cell wall expansion. Any accumulated formaldehyde is rapidly bound to tetrahydrofolate or coupled to nucleophiles such as glutathione (GSH) resulting in S-hydroxymethylglutathione which is subsequently utilised by a glutathione-NAD-dependent formaldehyde dehydrogenase to form S-formylglutathione. The aim of this study was to examine the little understood pathway of formaldehyde detoxification in Arabidopsis thaliana, by cloning and characterising the key esterase S-formylglutathione hydrolase (FGH). The activity of FGH crucially recycles glutathione and renders formate available to the C1 pathway. Previously identified in bacteria, yeast and humans FGH from A. thaliana was cloned by RT-PCR, with a translated open reading frame that consisted of 284 amino acids and showed appreciable similarity with human esterase D. Over-expression using a pET vector system in combination with Escherichia coli resulted in a protein 30 kDa in size as determined by SDS-PAGE. Soluble A. thaliana FGH extracted from E. coli demonstrated a clear affinity for S-formylglutathione ( K m 0.318 mM) and was inhibited, 48% and 59%, respectively, by the addition of 1 μm 5,5′-dithio-bis (2-nitrobenzoic acid) (DTNB) and p-hydroxy-mercuribenzoic acid (PHMB) indicating that an SH group may be essential for hydrolytic activity. The activity of S-formylglutathione hydrolase in the leaves of A. thaliana demonstrated that this enzyme might be part of a universal detoxification pathway shared by a variety of organisms.