CRYSTALLOGRAPHIC STUDIES ON SHIKIMATE KINASE FROM ERWINIA CHRYSANTHEMI. A.J. Lapthorn, T. Krell, J.E. Coyle and J.R. Coggins Dept.'s of Chemistry and Biochemistry, University of Glasgow, Glasgow, G12 8QQ, UK
Shikimate kinase (EC 2.7.1.71) catalyses the fifth reaction of the shikimate pathway which involves the conversion of shikimic acid into its phosphorylated derivative, shikimate 3-phosphate, using ATP as a co-substrate. The shikimate pathway is essential to plants and microorganisms for the biosynthesis of aromatic compounds, but importantly is absent from animals. The enzymes are therefore attractive targets for the development of novel antibiotics and herbicides such as glyphosate and fluroshikimate.
Crystals of an ADP-shikimic acid complex of Erwinia chrysanthemi shikimate kinase expressed in E. Coli have been obtained at room temperature using 0.1M HEPES pH=6.9 and 2.2M sodium chloride as precipitant. The crystals are tetragonal, space group P41212 or enantiomorph, a=108.5Å; c=92.8Å; with 2 molecules in the asymmetric unit, corresponding to a packing density of 3.6Å3Da-1. The crystals diffract to at least 2.6Å at the Daresbury SRS, station 9.5.
We anticipate that shikimate kinase will contain a core structure of 4 [[beta]]-strands and 4 [[alpha]]-helices as proposed for a number of ATP/GTP-binding proteins [1]. Matsuo and Nishikawa [2] have predicted that shikimate kinase has the same structure as adenylate kinase, but molecular replacement with AmoRe using this structure, and other kinases as search model have been unsuccessful. We are presently using MIR methods to solve the structure.
[1] Milner-White, J.E., Coggins, J.R and Anton, I.A. (1991) J.Mol. Biol. 221,751-754
[2] Matsuo, Y. ad Nishikawa, K. (1994) Prot. Sci. 11, 2055-2063.