The l-Cysteine/l-Cystine Shuttle System Provides Reducing Equivalents to the Periplasm in Escherichia coli

Ohtsu, Iwao; Wiriyathanawudhiwong, Natthawut; Morigasaki, Susumu; Nakatani, Takeshi; Kadokura, Hiroshi; Takagi, Hiroshi

doi:10.1074/jbc.m109.081356

Cited by 111 publications

(102 citation statements)

References 34 publications

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“…Because cysteine changed the cellular redox state, leading to greatly increased glutathione disulfide, it was possible that this change also increased the yield of products. Cysteine recently was shown to oxidize the E. coli cytoplasm (28). Using metabolomics and redox-sensitive GFP, we confirmed that cysteine greatly oxidizes E. coli ( Fig.…”

Section: Resultssupporting

confidence: 53%

Metabolic model for diversity-generating biosynthesis

Tianero

Pierce

Raghuraman

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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A conventional metabolic pathway leads to a specific product. In stark contrast, there are diversity-generating metabolic pathways that naturally produce different chemicals, sometimes of great diversity. We demonstrate that for one such pathway, tru, each ensuing metabolic step is slower, in parallel with the increasing potential chemical divergence generated as the pathway proceeds. Intermediates are long lived and accumulate progressively, in contrast with conventional metabolic pathways, in which the first step is rate-limiting and metabolic intermediates are shortlived. Understanding these fundamental differences enables several different practical applications, such as combinatorial biosynthesis, some of which we demonstrate here. We propose that these principles may provide a unifying framework underlying diversity-generating metabolism in many different biosynthetic pathways.natural products | secondary metabolism | biosynthesis | RiPP | cyanobactin T here are two fundamentally different types of metabolic pathways in living systems. The first are aimed to generate one or a few discrete chemicals; these comprise the majority of pathways. The second have evolved to produce large numbers of different metabolites (1, 2). Although perhaps fewer in number, this second class, which we will call "diversity generating" (DG), may be responsible for the majority of small molecules in living systems. A key difference is that the compounds produced in the latter generally have a more limited phylogenetic distribution.The metabolic pathways first elucidated were for the synthesis of essential metabolites found in all cells, such as amino acids, purines, or pyrimidines. These conventional metabolic pathways typically comprise multiple metabolic steps, with the intermediates generated in each step converted only to the final product of the pathway. DG pathways, however, do not yield a single final product. Each enzyme in a DG pathway has relaxed substrate specificity and is able to handle a variety of compounds, carrying out the same chemical transformation on different substrates.Previously, an evolutionary framework was developed to explain why some biosynthetic pathways produce many compounds (3-5). In this study we provide the first integrated overview, to our knowledge, of the multiple metabolic steps that comprise a DG biosynthetic pathway. We have uncovered striking differences in how this pathway differs from the canonical features of conventional pathways. Our results provide an initial framework for understanding how DG pathways are designed and how key features of such pathways diverge from the textbook model.We specifically examine the tru and related pat cyanobactin pathways (1, 6). These ribosomally synthesized and posttranslationally modified (RiPP) secondary metabolite pathways were identified in cyanobacterial symbionts of coral reef animals. Their expression required transfer to a model host, Escherichia coli (Fig.

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Section: Resultssupporting

confidence: 53%

Metabolic model for diversity-generating biosynthesis

Tianero

Pierce

Raghuraman

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…EamA and EamB, the representative cysteine exporters of E. coli (13,14), may serve primarily as components of the cysteine/ cystine shuttle system that protects cells from ROS. Moreover, these exporters are highly induced by hydrogen peroxide compared with cysteine, suggesting that their physiological function is to protect cells against ROS (18). CydDC transports cysteine and glutathione (15,17) and mediates cytochrome assembly (47).…”

Section: Discussionmentioning

confidence: 99%

“…However, the specificity of these transporters for cysteine and their significance in controlling cysteine levels as a safety valve are unclear. For example, CydDC and Bcr are primarily transporters of glutathione and multiple drugs (e.g., bicyclomycin), respectively (16,17), and EamA and EamB may function primarily as protection against reactive oxygen species (ROS) (18). Here, we describe two novel systems of Pantoea ananatis, a member of the family Enterobacteriaceae (19), which are directly involved in resistance to excess levels of cysteine through mecha-nisms that mediate cysteine degradation and efflux.…”

mentioning

confidence: 99%

Bacterial Cysteine-Inducible Cysteine Resistance Systems

Takumi

Nonaka

2016

J Bacteriol

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Cysteine donates sulfur to macromolecules and occurs naturally in many proteins. Because low concentrations of cysteine are cytotoxic, its intracellular concentration is stringently controlled. In bacteria, cysteine biosynthesis is regulated by feedback inhibition of the activities of serine acetyltransferase (SAT) and 3-phosphoglycerate dehydrogenase (3-PGDH) and is also regulated at the transcriptional level by inducing the cysteine regulon using the master regulator CysB. Here, we describe two novel cysteine-inducible systems that regulate the cysteine resistance of Pantoea ananatis, a member of the family Enterobacteriaceae that shows great potential for producing substances useful for biotechnological, medical, and industrial purposes. One locus, designated ccdA (formerly PAJ_0331), encodes a novel cysteine-inducible cysteine desulfhydrase (CD) that degrades cysteine, and its expression is controlled by the transcriptional regulator encoded by ccdR (formerly PAJ_0332 or ybaO), located just upstream of ccdA. The other locus, designated cefA (formerly PAJ_3026), encodes a novel cysteine-inducible cysteine efflux pump that is controlled by the transcriptional regulator cefR (formerly PAJ_3027), located just upstream of cefA. To our knowledge, this is the first example where the expression of CD and an efflux pump is regulated in response to cysteine and is directly involved in imparting resistance to excess levels of cysteine. We propose that ccdA and cefA function as safety valves that maintain homeostasis when the intra-or extracellular cysteine concentration fluctuates. Our findings contribute important insights into optimizing the production of cysteine and related biomaterials by P. ananatis. IMPORTANCEBecause of its toxicity, the bacterial intracellular cysteine level is stringently regulated at biosynthesis. This work describes the identification and characterization of two novel cysteine-inducible systems that regulate, through degradation and efflux, the cysteine resistance of Pantoea ananatis, a member of the family Enterobacteriaceae that shows great potential for producing substances useful for industrial purposes. We propose that this novel mechanism for sensing and regulating cysteine levels is a safety valve enabling adaptation to sudden changes in intra-or extracellular cysteine levels in bacteria. Our findings provide important insights into optimizing the production of cysteine and related biomaterials by P. ananatis and also a deep understanding of sulfur/cysteine metabolism and regulation in this plant pathogen and related bacteria.

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“…SiR activity was estimated by the coupled SiR/OAS-TL assay (Bosma et al, 1991;Khan et al, 2010) with the addition of NADPH and tungstic acid (Brychkova et al, 2012c). The resultant generated Cys was detected as described before (Gaitonde, 1967;Burandt et al, 2001;Ohtsu et al, 2010;Brychkova et al, 2012c). SiR in gel activity detection is based on the detection of sulfide, the direct product of SiR activity, reacting with lead acetate to yield lead sulfide bands (Brychkova et al, 2012c).…”

Section: Protein Extraction and Kinetic Assays For So Apr And Sir Amentioning

confidence: 99%

An Essential Role for Tomato Sulfite Oxidase and Enzymes of the Sulfite Network in Maintaining Leaf Sulfite Homeostasis

et al. 2012

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Little is known about the homeostasis of sulfite levels, a cytotoxic by-product of plant sulfur turnover. By employing extended dark to induce catabolic pathways, we followed key elements of the sulfite network enzymes that include adenosine-59-phosphosulfate reductase and the sulfite scavengers sulfite oxidase (SO), sulfite reductase, UDP-sulfoquinovose synthase, and b-mercaptopyruvate sulfurtransferases. During extended dark, SO was enhanced in tomato (Solanum lycopersicum) wild-type leaves, while the other sulfite network components were down-regulated. SO RNA interference plants lacking SO activity accumulated sulfite, resulting in leaf damage and mortality. Exogenous sulfite application induced up-regulation of the sulfite scavenger activities in dark-stressed or unstressed wild-type plants, while expression of the sulfite producer, adenosine-59-phosphosulfate reductase, was down-regulated. Unstressed or dark-stressed wild-type plants were resistant to sulfite applications, but SO RNA interference plants showed sensitivity and overaccumulation of sulfite. Hence, under extended dark stress, SO activity is necessary to cope with rising endogenous sulfite levels. However, under nonstressed conditions, the sulfite network can control sulfite levels in the absence of SO activity. The novel evidence provided by the synchronous darkinduced turnover of sulfur-containing compounds, augmented by exogenous sulfite applications, underlines the role of SO and other sulfite network components in maintaining sulfite homeostasis, where sulfite appears to act as an orchestrating signal molecule.

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The l-Cysteine/l-Cystine Shuttle System Provides Reducing Equivalents to the Periplasm in Escherichia coli

Cited by 111 publications

References 34 publications

Metabolic model for diversity-generating biosynthesis

Metabolic model for diversity-generating biosynthesis

Bacterial Cysteine-Inducible Cysteine Resistance Systems

An Essential Role for Tomato Sulfite Oxidase and Enzymes of the Sulfite Network in Maintaining Leaf Sulfite Homeostasis

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