Effect of oxidative stress on the biosynthesis of 2-C-methyl- d -erythritol-2,4-cyclopyrophosphate and isoprenoids by several bacterial strains

237

188

In many microorganisms, the putative orthologs of the Escherichia coli ygbB gene are tightly linked or fused to putative orthologs of ygbP, which has been shown earlier to be involved in terpenoid biosynthesis. The ygbB gene of E. coli was expressed in a recombinant E. coli strain and was shown to direct the synthesis of a soluble, 17-kDa polypeptide. The recombinant protein was found to convert 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate into 2C-methyl-D-erythritol 2,4-cyclodiphosphate and CMP. The structure of the reaction product was established by NMR spectroscopy using 13 C-labeled substrate samples. The enzyme-catalyzed reaction requires Mn 2؉ or Mg 2؉ but no other cofactors. Radioactivity from [2-14 C]2C-methyl-D-erythritol 2,4-cyclodiphosphate was diverted efficiently to carotenoids by isolated chromoplasts from Capsicum annuum and, thus, was established as an intermediate in the deoxyxylulose phosphate pathway of isoprenoid biosynthesis. YgbB protein also was found to convert 4-diphosphocytidyl-2C-methyl-D-erythritol into 2C-methyl-D-erythritol 3,4-cyclophosphate. This compound does not serve as substrate for the formation of carotenoids by isolated chromoplasts and is assumed to be an in vitro product without metabolic relevance.

Section: Resultssupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Biosynthesis of terpenoids: YgbB protein converts 4-diphosphocytidyl-2C-methyl- d -erythritol 2-phosphate to 2C-methyl- d -erythritol 2,4-cyclodiphosphate

Herz

Wungsintaweekul

Schuhr

et al. 2000

237

188

“…6 is a radical process akin to the one exploited by ribonucleotide reductase (35), in which the Fig. 4.…”

Section: Discussionmentioning

confidence: 99%

“…No clues are yet available concerning the nature of the reducing agent. An unusual sensitivity of the latter may explain why 2C-methyl-D-erythritol 2,4-cyclodiphosphate is accumulated in certain bacteria under conditions of oxidative stress (35).…”

Section: Discussionmentioning

confidence: 99%

Studies on the nonmevalonate pathway to terpenes: The role of the GcpE (IspG) protein

Hecht

Eisenreich

Adam

et al. 2001

194

153

Recombinant Escherichia coli cells engineered for the expression of the xylB gene in conjunction with genes of the nonmevalonate pathway were supplied with 13 C-labeled 1-deoxy-D-xylulose. Cell extracts were analyzed directly by NMR spectroscopy. 13 C-labeled 2C-methyl-D-erythritol 2,4-cyclodiphosphate was detected at high levels in cells expressing xylB, ispC, ispD, ispE, and ispF. The additional expression of the gcpE gene afforded 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate as an intermediate of the nonmevalonate pathway. Hypothetical mechanisms involving conserved cysteine residues are proposed for the enzymatic conversion of 2C-methyl-D-erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate catalyzed by the GcpE protein.isoprenoid biosynthesis ͉ 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate

“…Because both C. trachomatis and E. coli possess this pathway, it is possible that molecules downstream of the CT804 product are produced in increased quantities as a result of CT804 overexpression. Indeed, the subsequent product, MEC, is accumulated to high concentrations in several bacteria under conditions of stress (28). To determine which molecule is actually responsible for nucleoid decondensation, we partially reconstructed the MEP pathway by sequentially adding the expressed and partially purified orthologous chlamydial proteins, IspD (CT462), IspE (CT804), and IspF (CT434) to commercially available MEP.…”

Section: A Small Metabolite Is Required For In Vitro Nucleoid Decondementioning

confidence: 99%

Chlamydial histone-DNA interactions are disrupted by a metabolite in the methylerythritol phosphate pathway of isoprenoid biosynthesis

Grieshaber

Fischer

Mead

et al. 2004

The chlamydial developmental cycle is characterized by an intracellular replicative form, termed the reticulate body, and an extracellular form called the elementary body. Elementary bodies are characterized by a condensed chromatin, which is maintained by a histone H1-like protein, Hc1. Differentiation of elementary bodies to reticulate bodies is accompanied by dispersal of the chromatin as chlamydiae become transcriptionally active, although the mechanisms of Hc1 release from DNA have remained unknown. Dissociation of the nucleoid requires chlamydial transcription and translation with negligible loss of Hc1. A genetic screen was therefore designed to identify chlamydial genes rescuing Escherichia coli from the lethal effects of Hc1 overexpression. CT804, a gene homologous to ispE, which encodes an intermediate enzyme of the non-mevalonate methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis, was selected. E. coli coexpressing CT804 and Hc1 grew normally, although they expressed Hc1 to a level equivalent to that which condensed the chromatin of parent Hc1-expressing controls. Inhibition of the MEP pathway with fosmidomycin abolished IspE rescue of Hc1-expressing E. coli. Deproteinated extract from IspE-expressing bacteria caused dispersal of purified chlamydial nucleoids, suggesting that chlamydial histone-DNA interactions are disrupted by a small metabolite within the MEP pathway rather than by direct action of IspE. By partial reconstruction of the MEP pathway, we determined that 2-C-methylerythritol 2,4-cyclodiphosphate dissociated Hc1 from chlamydial chromatin. These results suggest that chlamydial histone-DNA interactions are disrupted upon germination by a small metabolite in the MEP pathway of isoprenoid biosynthesis.C hlamydia trachomatis is the leading cause of preventable blindness worldwide and a major cause of sexually transmitted disease in developed countries (1). Chlamydiae are bacterial obligate intracellular pathogens with a biphasic developmental cycle that alternates between infectious extracellular forms, termed elementary bodies (EBs), and intracellular replicative forms known as reticulate bodies (RBs) (2). The metabolically inert EBs are characterized by a condensed nucleoid structure. Within a few hours after infection the chromatin becomes dispersed as transcription is initiated and differentiation to the larger, metabolically active RBs begins (3). RBs replicate by binary fission until 18 to 48 h after infection, at which time they begin to differentiate back to EBs, a process typified by recompaction of the nucleoid.The DNA of EBs is held in a condensed state by the action of two histone H1 homologs, Hc1 and Hc2 (4-8). Hc1 is conserved among all chlamydiae, whereas Hc2 displays a variable molecular weight depending on the C. trachomatis serovar and is absent from some chlamydial species and strains (5). Both hctA and hctB, encoding Hc1 and Hc2, respectively, are transcribed late in the developmental cycle concomitant with RB differentiation back to EBs and nucleoid cond...