Verlag des Forschungszentrums Jülich
JUEL-3799
Grolle, Sigrid
Untersuchungen zu einem neuen Isopentenyldiphosphat-Biosyntheseweg in Escherichia coli und Zymomonas mobilis: Identifizierung und Charakterisierung beteiligter Gene
111 S., 2000
In the last few years, evidence has emerged that in bacteria a novel biosynthetic pathway to isopentenyl diphosphate exists. In this pathway isopentenyl diphosphate is formed from pyruvate and glyceraldehyde 3-phosphate. In the first reaction step these C3 compounds are combined to 1-deoxyxylulose 5-phosphate whereby pyruvate is decarboxylated. The gene encoding the 1-deoxyxylulose 5-phosphate synthase was identified in E. coli by searching for transketolase-homologous genes. The corresponding gene product was purified and was shown by NMR analysis to catalyze in a thiamin diphosphate (TPP) and Mg2+ dependent reaction the synthesis of 1-deoxyxylulose 5-phosphate. Phylogenetic investigation revealed that the 1-deoxyxylulose 5-phosphate synthase belongs to a new family of TPP dependent enzymes. The dxs gene is located at 9 min on the E. coli chromosome and is organized in one operon with a putative aldoketo-reductase gene. The disruption of this E. coli gene yielded no phenotype which indicates that the gene is not involved in the novel pathway. The gene encoding the second enzyme of the pathway, the 1-deoxyxylulose 5-phosphate reductoisomerase, was isolated from Z. mobilis. The 1-deoxyxylulose 5-phosphate reductoisomerase catalyzes the NADPH and Mn2+ dependent rearrangement and subsequent reduction of 1-deoxyxylulose 5-phosphate to 2C-methylerythritol 4-phosphate. The enzyme activity is competitively inhibited by the antibiotic fosmidomycin with a Ki of 0,6 µM.
By using a E. coli strain engineered to produce the isoprenoide zeaxanthin the gene encoding IPP-isomerase, but no further genes of the novel pathway could be isolated from an E. coli expression gene library.
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