title: Heterologous expression, purification, crystallization and preliminary X-ray diffraction analysis of KDO8P synthase from Arabidopsis thaliana.
abstract: 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.
authors.affiliation: The Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, Lin'an, Zhejiang 311300, China.
authors.forename: Fengxue
authors.initials: F
authors.lastname: Zhang
authors.affiliation: The Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, Lin'an, Zhejiang 311300, China.
authors.forename: Yingwu
authors.initials: Y
authors.lastname: Xu
authors.affiliation: The Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, Lin'an, Zhejiang 311300, China. Electronic address: 397942805@qq.com.
authors.forename: Zhijun
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D000446:Aldehyde-Lyases / Q000096:biosynthesis / Q000235:genetics* / Q000648:ultrastructure*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D002850:Chromatography, Gel; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: C020011:2-dehydro-3-deoxyphosphooctonate aldolase; D000446:Aldehyde-Lyases
keywords: Analytical ultracentrifuge; Arabidopsis thaliana; Crystallization; KDO8P synthase; Purification; X-ray