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| Home > Publications database > 1-Desoxy-D-xylulose-5 Phosphat Synthase von Escherichia coli : biochemische Charakterisierung und Struktur-Funktions-Beziehungen |
| Home > Publications database > 1-Desoxy-D-xylulose-5 Phosphat Synthase von Escherichia coli : biochemische Charakterisierung und Struktur-Funktions-Beziehungen |
| Dissertation / PhD Thesis/Book | PreJuSER-27352 |
2001
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
Please use a persistent id in citations: http://hdl.handle.net/2128/20003
Report No.: Juel-3904
Abstract: 1-deoxy-D-xylulose 5-phosphate synthase (DXS) from Escherichia coli catalyses the committed enzymatic step in the biosynthesis of thiamin, pyridoxal and isopentenyl diphosphate in a thiamin diphosphate (ThDP) dependent acyloin condensation of pyruvate and D-glyceraldehyde 3-phosphate (D-GAP). The aim of this work was to investigate the biochemical properties and the relationship of structure and function of DXS from E. coli as the first member of a new family of ThDP enzymes. Furthermore it was intended to analyse the possibility to employ the enzyme in chemo-enzymatic synthesis, since enzymes with ThDP as a cofactor have proven to be versatile tools in the organic synthesis of chiral building blocks and natural products . DXS was purified to apparent homogeneity as a fusion protein containing a carboxy-terminal his-tag at high yields . The enzyme showed a broad pHspectrum ranging from pH 5.5 to 10.5 and a half-life of more than 47 days at 4°C in a Hepes buffer. DXS is reacting with a ping pong mechanism and has a high affinity towards its cofactors ThDP (KM 0.3 pM) and Mg2+ (KM < 10 NM). The conversion of the physiological substrates pyruvate and D-GAP is catalysed with 4.3 U/mg protein and KM-values of 0.10 and 0.44 mM by DXS, respectively . The enzyme could be used for the racemic resolution of D,L-glyceraldehyde, since only the D-form was converted with a specific activity of 4.3 U/mg and a KM value of 10.4 mM. Hydroxypyruvate and aketobutyrate are competitive inhibitors of DXS with K; values of 2 .4 mM and 6.5 mM, respectively. Moreover they are alternative donor substrates with respect to pyruvate, which are converted with 0.3 U/mg and 0.1 U/mg, respectively. Short-chain aldehydes and aldoses from C2-C5 like glycolaldehyde and D-erythrose 4-phosphate served as alternative acceptor substrates and are converted at high rates of 71% and 84% respectively compared to D-glyceraldehyde . The complete conversion of pyruvate and D-ribose 5-phosphate with DXS yielded 1-deoxy-D-sedoheptulose 7-phosphate . A disaccharide of glucose and 1-deoxy-D-xylulose was synthesised in a coupled enzymatic reaction with DXS and sucrose synthase from Solanum tuberosum. The DXS from E. coli constitutes a homodimer of 142 kDa and shows similarities in domain structure and substrate binding to transketolases . A working model of the substrate channel of DXS was built comparing DXS and transketolase sequences with respect to known transketolase structures and verified by site-directed mutagenesis. GIu370 is responsible for the activation of ThDP and Phel07 for donor substrate binding . The histidine residues 49 and 257 are involved in catalysis, while Asp427 interacts with the C2-OH of the acceptor substrate. Arg478 complexes the phosphate group of acceptor substrates. Unphosphorylated substrates are converted with higher efficiency by a Arg478G1n mutein compared to wildtype DXS.
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