2-Deoxy-4- epi- scyllo-inosose (DEI) is the Product of EboD, a Highly Conserved Dehydroquinate Synthase-like Enzyme in Bacteria and Eustigmatophyte Algae

Abstract

A cryptic cluster of genes, known as the ebo cluster, has been found in a variety of genomic contexts among bacteria and algae. In Pseudomonas fluorescens NZI7, the ebo cluster (a.k.a. EDB cluster) is involved in the bacterial repellent mechanism against nematode grazing. In cyanobacteria, the cluster plays a role in the transport of the scytonemin monomer from the cytosol to the periplasm. Despite their broad distribution and interesting phenotypes, neither the pathway nor the functions of the enzymes are known. Here we show that EboD proteins from the ebo clusters in Nostoc punctiforme and Sporocytophaga myxococcoides catalyze the cyclization of mannose 6-phosphate to a novel cyclitol, 2-deoxy-4-epi-scyllo-inosose. The enzyme product is postulated to be a precursor of a signaling molecule or a transporter in the organisms. This study sheds the first light onto ebo/EDB pathways and established a functionally distinct enzyme that extends the diversity of sugar phosphate cyclases.

Figure 1.

Distribution of DHQS and related SPCs found in the NCBI database. (a) Phylogenetic tree of DHQS and DHQS homologues; (b) The numbers of DHQS homologues separated on the basis of their putative functions; (c) Ebo and EDB gene clusters in a number of bacteria.

Figure 2.

Biochemical characterization of recombinant EboD. (a–b) Chemical structures of C₇- and C₆-sugar phosphates investigated as potential substrates for EboD; (c) TLC analysis of EboD reactions with various C₆-sugar phosphates; (d) Schematic conversion of Man6P to DEI by EboD; (e) COSY and selected HMBC and NOESY correlations of DEI; and (f) Extracted ion chromatogram of EboD reaction with Man6P.

Figure 3.

Proposed structure and catalytic mechanism of EboD. (a) Unique active site residues for EboD and other SPCs that may be involved in their distinct substrate recognition; (b) comparison of the sugar binding site for DOIS (green) and EboD (grey). The red star points to the C-4 hydroxyl for the dephosphorylated carbaglucose-6-phosphate inhibitor (yellow) from the DOIS structure (PDB 2GRU). Oxygens are shown in red, nitrogens in blue and a water molecule as a red sphere. Distances are represented in Angstroms; (c) proposed catalytic mechanism of EboD, which catalyzes the cyclization of Man6P to DEI.

Scheme 1.

Sugar phosphate cyclases involved in primary and secondary metabolism. DHQS, 3-dehydroquinate synthase; aminoDHQS, 5-deoxy-5-amino-3-dehydroquinate synthase; DOIS, 2-deoxy-scyllo-inosose synthase; EEVS, 2-epi-5-epi-valiolone synthase; EVS, 2-epi-valiolone synthase; DDGS, 2-desmethyl-4-deoxy-gadusol synthase; and EboD, 2-deoxy-4-epi-scyllo-inosose (DEI) synthase.