Functional and biochemical characterization of a recombinant Arabidopsis thaliana 3-deoxy-D-manno-octulosonate 8-phosphate synthase.
An open reading frame, encoding for KDOPS (3-deoxy-D-manno-octulosonate 8-phosphate synthase), from Arabidopsis thaliana was cloned into a T7-driven expression vector. The protein was overexpressed in Escherichia coli and purified to homogeneity. Recombinant A. thaliana KDOPS, in solution, displays an apparent molecular mass of 76 kDa and a subunit molecular mass of 31.519 kDa. Unlike previously studied bacterial KDOPSs, which are tetrameric, A. thaliana KDOPS appears to be a dimer in solution. The optimum temperature of the enzyme is 65 degrees C and the optimum pH is 7.5, with a broad peak between pH 6.5 and 9.5 showing 90% of maximum activity. The enzyme cannot be inactivated by EDTA or dipicolinic acid treatment, nor it can be activated by a series of bivalent metal ions, suggesting that it is a non-metallo-enzyme, as opposed to the initial prediction that it would be a metallo-enzyme. Kinetic studies showed that the enzyme follows a sequential mechanism with K(m)=3.6 microM for phosphoenolpyruvate and 3.8 microM for D-arabinose 5-phosphate and kcat=5.9 s(-1) at 37 degrees C. On the basis of the characterization of A. thaliana KDOPS and phylogenetic analysis, plant KDOPSs may represent a new, distinct class of KDOPSs.
Wu
Jing
J
Department of Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109-1065, USA.
Patel
Mayur A
MA
Sundaram
Appavu K
AK
Woodard
Ronald W
RW
D010723:3-Deoxy-7-Phosphoheptulonate Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010940:Plant Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment / Q000379:methods; D013379:Substrate Specificity; D013400:Sugar Acids; D013403:Sugar Phosphates / Q000096:biosynthesis; D013696:Temperature
C121801:3-deoxy-manno-octulosonate-8-phosphate; D029681:Arabidopsis Proteins; D001426:Bacterial Proteins; D008670:Metals; D010940:Plant Proteins; D011994:Recombinant Proteins; D013400:Sugar Acids; D013403:Sugar Phosphates; D010723:3-Deoxy-7-Phosphoheptulonate Synthase