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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Structure and Properties of an Engineered Transketolase from Maize¹

Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, D–85747 Garching, Germany (S.G., S.E., A.B., M.F.); Bayer CropScience AG, Research-Target Research, Building 6240, Alfred-Nobel-Strasse 50, D–40789 Monheim, Germany (M.B., J.F., G.A.), Max-Planck-Institut für Biochemie, Abteilung Strukturforschung, Am Klopferspitz 18a, D–82152 Martinsried, Germany (G.B., R.H.); Institut für Genetik, Technische Universität Braunschweig, Spielmannstrasse 7, D–38106 Braunschweig, Germany (W.F.M.)

The gene specifying plastid transketolase (TK) of maize (Zea mays) was cloned from a cDNA library by southern blotting using a heterologous probe from sorghum (Sorghum bicolor). A recombinant fusion protein comprising thioredoxin of Escherichia coli and mature TK of maize was expressed at a high level in E. coli and cleaved with thrombin, affording plastid TK. The protein in complex with thiamine pyrophoshate was crystallized, and its structure was solved by molecular replacement. The enzyme is a C2 symmetric homodimer closely similar to the enzyme from yeast (Saccharomyces cerevisiae). Each subunit is folded into three domains. The two topologically equivalent active sites are located in the subunit interface region and resemble those of the yeast enzyme.

¹ This work was supported by the Deutsche Forschungsgemein-schaft and by the Fonds der Chemischen Industrie.

² These authors contributed equally to the paper.

³ Present address: AstraZeneca, Alderley Park, Macclesfield SK10 4TG, UK.

⁴ Present address: Bayer BioScience N.V., Technology Discovery, Jozef Plateaustraat 22, B–9000 Gent, Belgium.

⁵ Present address: Antisense Pharma GmbH, Josef-Engert-Strasse 9, D–93053 Regensburg, Germany.

⁶ Present address: Institut für Botanik III, Universität Düsseldorf, Universitätsstr. 1, D–40225 Düsseldorf, Germany.

^* Corresponding author; e-mail markus.fischer{at}ch.tum.de; fax 49–89–289–13363.

Received January 24, 2003; returned for revision March 4, 2003; accepted May 6, 2003.