Characterization of two 3-deoxy-d-Arabino-Heptulosonate 7-phosphate synthases from Bacillusmethanolicus

Abstract

3-Deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with d-erythrose 4-phosphate (E4P) and plays an important role in regulating carbon flux toward aromatic amino acid biosynthesis in bacteria and plants. Sequence analysis of the DAHP synthases AroG1 and AroG2 from Bacillus methanolicus MGA3 suggested this thermophilic, methylotrophic bacterium possesses two type Iβ DAHP synthases. This study describes production of AroG1 and AroG2 in Escherichia coli as hexa-histidine fused proteins, which were purified by affinity chromatography. Treatment with TEV protease afforded native proteins for characterization and kinetic analysis. AroG1 and AroG2 are, respectively, 30.1 kDa and 40.0 kDa proteins. Both enzymes have maximal activity over a pH range of 6.3-7.2. The apparent kinetic parameters at 50 °C and pH 7.2 for AroG1 are K_m^PEP 1100 ± 100 μM, K_m^E4P 530 ± 100 μM, and k_cat 10.3 ± 1.2 s^-1. The kinetic parameters for AroG2 are K_m^PEP 90 ± 20 μM, K_m^E4P 130 ± 40 μM, and k_cat 2.0 ± 0.2 s^-1. At 50 °C AroG2 retains 50% of its activity after 96 min whereas AroG1 retains less than 5% of its activity after 10 min. AroG2, which contains an N-terminal regulatory domain, is inhibited by chorismate and prephenate but not l-phenylalanine, l-tyrosine, or l-tryptophan. AroG1 is not inhibited by any of the molecules examined. Understanding DAHP synthase regulation in B. methanolicus is a first step toward generating biocatalysts that exploit the target-rich aromatic amino acid biosynthetic pathway for synthesis of chemicals from methanol.

Keywords: 3-Deoxy-d-arabino-heptulosonate 7-phosphate synthase; Aromatic amino acid biosynthesis; Bacillus methanolicus; Chorismate; Methylotrophy; Shikimic acid.