Dehydroquinate dehydratase/shikimate dehydrogenases involved in gallate biosynthesis of the aluminum-tolerant tree species Eucalyptus camaldulensis

Ko Tahara,Evelyn Funke,Carsten Milkowski,Takafumi Miyama,Shin-Ichi Miyazawa,Mitsuru Nishiguchi

doi:10.1007/s00425-020-03516-w

Abstract

Main conclusionEucalyptus camaldulensis EcDQD/SDH2 and 3 combine gallate formation, dehydroquinate dehydratase, and shikimate dehydrogenase activities. They are candidates for providing the essential gallate for the biosynthesis of the aluminum-detoxifying metabolite oenothein B.The tree species Eucalyptus camaldulensis shows exceptionally high tolerance against aluminum, a widespread toxic metal in acidic soils. In the roots of E. camaldulensis, aluminum is detoxified via the complexation with oenothein B, a hydrolyzable tannin. In our approach to elucidate the biosynthesis of oenothein B, we here report on the identification of E. camaldulensis enzymes that catalyze the formation of gallate, which is the phenolic constituent of hydrolyzable tannins. By systematical screening of E. camaldulensis dehydroquinate dehydratase/shikimate dehydrogenases (EcDQD/SDHs), we found two enzymes, EcDQD/SDH2 and 3, catalyzing the NADP+-dependent oxidation of 3-dehydroshikimate to produce gallate. Based on extensive in vitro assays using recombinant EcDQD/SDH2 and 3 enzymes, we present for the first time a detailed characterization of the enzymatic gallate formation activity, including the cofactor preferences, pH optima, and kinetic constants. Sequence analyses and structure modeling suggest the gallate formation activity of EcDQD/SDHs is based on the reorientation of 3-dehydroshikimate in the catalytic center, which facilitates the proton abstraction from the C5 position. Additionally, EcDQD/SDH2 and 3 maintain DQD and SDH activities, resulting in a 3-dehydroshikimate supply for gallate formation. In E. camaldulensis, EcDQD/SDH2 and 3 are co-expressed with UGT84A25a/b and UGT84A26a/b involved in hydrolyzable tannin biosynthesis. We further identified EcDQD/SDH1 as a “classical” bifunctional plant shikimate pathway enzyme and EcDQD/SDH4a/b as functional quinate dehydrogenases of the NAD+/NADH-dependent clade. Our data indicate that in E. camaldulensis the enzymes EcDQD/SDH2 and 3 provide the essential gallate for the biosynthesis of the aluminum-detoxifying metabolite oenothein B.

Highlights

In plants and microorganisms, the shikimate pathway produces chorismate, the metabolic precursor of aromatic amino acids, and is essential for protein biosynthesis
Because EcC015288.50 was represented by two closely related sequence variants, our homology-based RT-PCR cloning approach resulted in the isolation of five full-length candidate cDNAs from E. camaldulensis roots
They were designated as EcDQD/SDH1 (Accession No LC487988), EcDQD/SDH2 (LC487989), EcDQD/SDH3 (LC487990), EcDQD/SDH4a (LC487991), and EcDQD/ SDH4b (LC487992)

Summary

Introduction

The shikimate pathway produces chorismate, the metabolic precursor of aromatic amino acids, and is essential for protein biosynthesis. Plant SDH enzymes are fused to dehydroquinate dehydratases (DQDs, EC 4.2.1.10) to form bifunctional DQD/ SDH enzymes (Bischoff et al 2001; Peek and Christendat 2015). “classical” DQD/ SDH enzymes catalyze two successive reactions (Fig. 1), the dehydration of 3-dehydroquinate (3-DHQ) to 3-dehydroshikimate (3-DHS) (reaction 1, DQD activity) and the reversible reduction of 3-DHS to shikimate (reaction 2, SDH activity). Genome analyses revealed that many seed plants contain genes for multiple DQD/SDH enzymes (Carrington et al 2018 ; Huang et al 2019). In some of these plant species, diverse DQD/SDH functions were observed.

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