Abstract
The plant shikimate pathway directs a significant portion of photosynthetically assimilated carbon into the downstream biosynthetic pathways of aromatic amino acids (AAA) and aromatic natural products. 3-Deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase (hereafter DHS) catalyzes the first step of the shikimate pathway, playing a critical role in controlling the carbon flux from central carbon metabolism into the AAA biosynthesis. Previous biochemical studies suggested the presence of manganese- and cobalt-dependent DHS enzymes (DHS-Mn and DHS-Co, respectively) in various plant species. Unlike well-studied DHS-Mn, however, the identity of DHS-Co is still unknown. Here, we show that all three DHS isoforms of Arabidopsis thaliana exhibit both DHS-Mn and DHS-Co activities in vitro. A phylogenetic analysis of various DHS orthologs and related sequences showed that Arabidopsis 3-deoxy-D-manno-octulosonate-8-phosphate synthase (KDOPS) proteins were closely related to microbial Type I DHSs. Despite their sequence similarity, these Arabidopsis KDOPS proteins showed no DHS activity. Meanwhile, optimization of the DHS assay conditions led to the successful detection of DHS-Co activity from Arabidopsis DHS recombinant proteins. Compared with DHS-Mn, DHS-Co activity displayed the same redox dependency but distinct optimal pH and cofactor sensitivity. Our work provides biochemical evidence that the DHS isoforms of Arabidopsis possess DHS-Co activity.