Evolutionary divergence of sedoheptulose 7-phosphate cyclases leads to several distinct cyclic products

Abstract

Sedoheptulose 7-phosphate cyclases are enzymes that utilize the pentose phosphate pathway intermediate, sedoheptulose 7-phosphate, to generate cyclic precursors of many bioactive natural products, such as the antidiabetic drug acarbose, the crop protectant validamycin, and the natural sunscreens mycosporine-like amino acids. These proteins are phylogenetically related to the dehydroquinate (DHQ) synthases from the shikimate pathway and are part of the more recently recognized superfamily of sugar phosphate cyclases, which includes DHQ synthases, aminoDHQ synthases, and 2-deoxy-scyllo-inosose synthases. Through genome mining and biochemical studies, we identified yet another subset of DHQS-like proteins in the actinomycete Actinosynnema mirum and the myxobacterium Stigmatella aurantiaca DW4/3-1. These enzymes catalyze the conversion of sedoheptulose 7-phosphate to 2-epi-valiolone, which is predicted to be an alternative precursor for aminocyclitol biosynthesis. Comparative bioinformatics and biochemical analyses of these proteins with 2-epi-5-epi-valiolone synthases (EEVS) and desmethyl-4-deoxygadusol synthases (DDGS) provided further insights into their genetic diversity, conserved amino acid sequences, and plausible catalytic mechanisms. The results further highlight the uniquely diverse DHQS-like sugar phosphate cyclases, which may provide new tools for chemoenzymatic, stereospecific synthesis of various cyclic molecules.

Figure 1

Genome mining leading to the discovery of new genes that encode DHQ synthase-like proteins. The presence of DHQS genes (yellow arrows) together with the putative pseudoglycosyltransferase (Ps-GT) genes (orange arrows) in the clusters raised questions as to whether DHQS are involved in pseudosugar (cyclitol) formation.

Figure 2

Phylogenetic analysis of the superfamily of sugar phosphate cyclases. Boxed proteins are representatives of families of sugar phosphate cyclases being studied in this paper. Maximum likelihood analysis was carried out using MEGA5 software with a WAG amino acid substitution model and a discrete gamma distribution was used to model evolutionary rate differences among sites. The robustness of the trees was assessed by bootstrap analysis (100 replicates). The source and accession number of the proteins are listed in Table S1.

Figure 3

Characterization of Amir_2000, Staur_3140, and related enzymes. A, SDS-PAGE of Ni-NTA purified cyclases. B, TLC analysis of the cylases' products with DAHP as substrate. C, TLC analysis of the cyclases' products with sedoheptulose 7-phosphate as substrate. D, Chemical structures of 2-epi-5-epi-valiolone, 5-epi-valiolone, 2-epi-valiolone, valiolone, and desmethyl-4-deoxygadusol.

Figure 4

In situ ¹H NMR analyses of ValA (A), Amir_2000 (B), and Npun_5600 (C) reactions.

Figure 5

Superimposition of twenty low-energy structures of EEV (A), 5EV (B), 2EV (C), and V (D). The first two low-energy conformations of 2EV are shown in the ball-and-stick representation (E and F).

Scheme 1

Synthesis of 2-epi-valiolone

Scheme 2

Most substrates for DHQ synthase-like cyclases are directly derived from the pentose phosphate pathway. EEVS, EVS, and DDGS all utilize sedoheptulose 7-phosphate as substrate but yield three different cyclic products.

Scheme 3

Proposed catalytic mechanisms for EEVS, EVS, and DDGS.