Heterologous expression, purification, crystallization and preliminary X-ray diffraction analysis of KDO8P synthase...

3-Deoxy-d-manno-octulosonate 8-phosphate synthase (KDO8PS) [EC 4.1.2.16] is the first and rate-limiting enzyme in the 3-deoxy-d-manno-octulosonate (KDO) biosynthetic pathway. The enzyme is widely expressed in bacteria and plants. Their well conserved protein sequences imply a similar oligomeric arrangement. However, the reported size exclusion chromatrographic analysis suggested a species-dependent self-assembling. To clarify the discrepancy and explore the self-assembling property of KDO8PS, we expressed and purified the Arabidopsis enzyme in Escherichia coli system. The enzyme was highly purified using a two-step purification strategy including nickel affinity and size exclusion chromatography with an expected pH activity profile. The identity of the purified enzyme was confirmed by Western-blot and mass fingerprints. Further analysis by analytical ultracentrifugation indicated that both bacteria and Arabidopsis enzymes are homotetramer. Furthermore, the purified enzyme from the plant has been crystallized and a complete set of X-ray data to 2.1Å resolution has been collected.