Early steps of isoprenoid biosynthesis in Escherichia coli

Abstract

The incorporation of 2H- and 13C-labelled precursors into ubiquinone-8 (Uq-8) by strains of Escherichia coli was measured in order to define the pathway for the early steps in the biosynthesis of isoprenoids in these eubacteria. Cells grown with DL-[methyl-2H6]valine were found to label both the alpha-oxoisovaleric (‘alpha-ketoisovaleric’) acid alpha-oxoisohexanoic (‘alpha-ketoisocaproic’) acid, but not the Uq-8. Since these acids are required for the biosynthesis of isoprenoids by the acetolactate pathway, the operation of this pathway in the biosynthesis of Uq-8 is excluded. Cells grown with [1,2-13C2]acetate and non-labelled glucose readily incorporated 13C2 units into fatty acids, but failed to incorporate any label into the Uq-8. Cells grown with [U-13C6]glucose and non-labelled acetate, however, were found to label both the fatty acids and the Uq-8. Oxidative cleavage with periodate/permanganate of the Uq-8 isolated from cells grown with U-13C6-labelled glucose produced laevulinic acid, which was shown to be derived from two C2 units and one C1 unit of the labelled glucose by mass-spectral analysis of the 4,5-dihydro-6-methyl-2-phenylpyridazin-3(2H)-one derivative. The results of this work indicate that the C-2 and C-3 carbon unit of pyruvate, not acetyl-CoA, is the precursor to isopentenyl pyrophosphate (IPP) in these cells; however, the labelling pattern observed is consistent with the established acetoacetate pathway of isoprenoid biosynthesis. These data, coupled with the observed lack of inhibition of the growth of E. coli by mevinolin, a specific inhibitor of 3-hydroxy-3-methylglutaryl-CoA, can be best rationalized by the biosynthesis of IPP occurring in E. coli through a series of bound intermediates.

Mevalonate-independent methylerythritol phosphate pathway for isoprenoid biosynthesis. Elucidation and distribution

“…[10]. 13 C-Labeled acetate was incorporated into the prenyl moiety of antibiotics of mixed origin from several actinomyces, and the labeling pattern corresponded to the expected one resulting from the direct incorporation of acetate into the MVA pathway [11]. Other incorporation attempts led to inconclusive results, which could not be readily interpreted.…”

“…This makes them well suited for investigation by NMR spectroscopy after feeding of 13 C-labeled precursors. Feeding of 13 C-labeled acetate to the hopanoid producing bacteria Rhodopseudomonas palustris, Rhodopseudomonas acidophila, or Methylobacterium organophilum were performed with bacteria grown on a mineral medium only containing 13 C-labeled acetate as the only carbon and energy source [14]. Such experimental conditions ensured the incorporation of the labeled precursor into the hopanoids.…”

“…The few studies leading to positive results pointed to the presence of the MVA pathway. 14 C-and 13 C-labeled MVA were respectively incorporated into a sterol of the gliding bacterium Nannocystis exedens [9] or into the carotenoids of a "Flavobacterium" sp. [10].…”

“…Thus, the incorporation of 14 C-labeled acetate into the prenyl chain of ubiquinone from Escherichia coli resulted in a radioactivity distribution, which did not fit with the expected one from the MVA pathway, and led the authors to propose an alternative variant of this pathway via acetolactate [12]. 13 C-Labeled acetate was not incorporated into this prenyl chain of ubiquinone, when E. coli was grown on a culture medium containing glucose and amino acid-derived carbon sources, whereas pyruvate labeled two contiguous carbon atoms derived from C3 and C5 of DMAPP or IPP [13]. Such results were indeed the premises for the presence of an alternative route for the formation of isoprene units.…”

“…Interpretation was not obvious from the sole experiments performed with labeled acetate. The clues for the elucidation of this metabolic pathway were obtained after incorporation of glucose isotopomers with 13 C labeling either at C1, C2, C3, C5, or C6 into the hopanoids of Zymomonas mobilis [16]. Indeed, Z. mobilis only utilizes glucose as carbon source.…”