Structure and characterization of the 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase from Aeropyrum pernix

Abstract

The first enzyme in the shikimic acid biosynthetic pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS), varies significantly in size and complexity in the bacteria and plants that express it. The DAH7PS from the archaebacterium Aeropyrum pernix (DAH7PSAp) is among the smallest and least complex of the DAH7PS enzymes, leading to the hypothesis that DAH7PSAp would not be subject to feedback regulation by shikimic acid pathway products. We overexpressed DAH7PSAp in Escherichia coli, purified it, and characterized its enzymatic activity. We then solved its X-ray crystal structure with a divalent manganese ion and phosphoenolpyruvate bound (PDB ID: 1VS1). DAH7PSAp is a homodimeric metalloenzyme in solution. Its enzymatic activity increases dramatically above 60°C, with optimum activity at 95°C. Its pH optimum at 60°C is 5.7. DAH7PSAp follows Michaelis–Menten kinetics at 60°C, with a KM for erythrose 4-phosphate of 280μM, a KM for phosphoenolpyruvate of 891μM, and a kcat of 1.0s−1. None of the downstream products of the shikimate biosynthetic pathway we tested inhibited the activity of DAH7PSAp. The structure of DAH7PSAp is similar to the structures of DAH7PS from Thermatoga maritima (PDB ID: 3PG8) and Pyrococcus furiosus (PDB ID: 1ZCO), and is consistent with its designation as an unregulated DAH7PS.