Structural and biochemical studies of sulphotransferase 18 from Arabidopsis thaliana explain its substrate specificity and reaction mechanism

Felix Hirschmann,Jutta Papenbrock,Jonathan Wolf Mueller,Dietmar J Manstein,Florian Krause,Roman Fedorov,Igor Chizhov,Petra Baruch

doi:10.1038/s41598-017-04539-2

Abstract

Sulphotransferases are a diverse group of enzymes catalysing the transfer of a sulfuryl group from 3′-phosphoadenosine 5′-phosphosulphate (PAPS) to a broad range of secondary metabolites. They exist in all kingdoms of life. In Arabidopsis thaliana (L.) Heynh. twenty-two sulphotransferase (SOT) isoforms were identified. Three of those are involved in glucosinolate (Gl) biosynthesis, glycosylated sulphur-containing aldoximes containing chemically different side chains, whose break-down products are involved in stress response against herbivores, pathogens, and abiotic stress. To explain the differences in substrate specificity of desulpho (ds)-Gl SOTs and to understand the reaction mechanism of plant SOTs, we determined the first high-resolution crystal structure of the plant ds-Gl SOT AtSOT18 in complex with 3′-phosphoadenosine 5′-phosphate (PAP) alone and together with the Gl sinigrin. These new structural insights into the determination of substrate specificity were complemented by mutagenesis studies. The structure of AtSOT18 invigorates the similarity between plant and mammalian sulphotransferases, which illustrates the evolutionary conservation of this multifunctional enzyme family. We identified the essential residues for substrate binding and catalysis and demonstrated that the catalytic mechanism is conserved between human and plant enzymes. Our study indicates that the loop-gating mechanism is likely to be a source of the substrate specificity in plants.

Highlights

Sulphotransferases (SOTs or SULTs) (EC 2.8.2.-) can be found in all organisms analysed so far
In Arabidopsis thaliana the three SOTs AtSOT16, AtSOT17 and AtSOT18 exclusively catalyse the transfer of a sulfuryl group to different ds-Gls[11,12,13]
SOTs were mainly studied at a physiological level[10], and until now, only one plant SOT structure was published, where the three substrate-binding loops showed no electron density at all[17]

Summary

Introduction

Sulphotransferases (SOTs or SULTs) (EC 2.8.2.-) can be found in all organisms analysed so far They catalyse the transfer of a sulfuryl group from the co-substrate 3′-phospho- adenosine 5′-phosphosulphate (PAPS) to a hydroxyl group of various substrates. The role of plant SOTs in the sulphation of desulpho-glucosinolates (ds-Gl) is of particular interest (Fig. 1), as they are important secondary metabolites and their break-down products are involved in defence against herbivores, pathogens, and abiotic stress in the plant order Brassicales[2]. Gl degradation products play a role as flavour compounds from numerous cabbage, radish and mustard species. All three enzymes are localized in the cytoplasm and their expression pattern tested under several conditions is similar[11] These three AtSOTs share at least 72% sequence identity but differ remarkably in their substrate specificity. The molecular basis for the observed substrate specificity of ds-Gl SOTs is not well understood

Methods

Results

Conclusion