Structure and properties of an engineered transketolase from maize.
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.
Gerhardt
Stefan
S
Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany.
Echt
Stefanie
S
Busch
Marco
M
Freigang
Jörg
J
Auerbach
Günter
G
Bader
Gerd
G
Martin
William F
WF
Bacher
Adelbert
A
Huber
Robert
R
Fischer
Markus
M
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D011487:Protein Conformation; D015202:Protein Engineering; D021122:Protein Subunits / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013835:Thiamine Pyrophosphate / Q000378:metabolism; D014174:Transketolase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics
D003067:Coenzymes; D021122:Protein Subunits; D014174:Transketolase; D013835:Thiamine Pyrophosphate