Crotonobetaine reductase from Escherichia coli consists of two proteins

Preußer, Andrea; Wagner, Ulrich; Elßner, Thomas; Kleber, Hans‐Peter

doi:10.1016/s0167-4838(99)00032-1

Cited by 37 publications

(70 citation statements)

References 34 publications

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“…In Escherichia coli, this pathway, which includes the dehydration and reduction of L-carnitine to g-butyrobetaine, is induced during anaerobic growth. The carnitine pathway has also been found to generate the osmoprotectant betaine during anaerobic respiration (Kleber, 1997;Preusser et al, 1999;Elssner et al, 2001). Recent analysis of the anaerobic carnitine reduction in E. coli has also shown that the electron transfer of flavoproteins FixA and FixB are necessary for the transfer of electrons to crotonobetaine reductase (CaiA) (Walt and Kahn, 2002) and we found FixA expressed in the sponge community (Supplementary Table S1).…”

Section: Metabolismsupporting

confidence: 52%

Metaproteogenomic analysis of a community of sponge symbionts

et al. 2012

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Sponges harbour complex communities of diverse microorganisms, which have been postulated to form intimate symbiotic relationships with their host. Here we unravel some of these interactions by characterising the functional features of the microbial community of the sponge Cymbastela concentrica through a combined metagenomic and metaproteomic approach. We discover the expression of specific transport functions for typical sponge metabolites (for example, halogenated aromatics, dipeptides), which indicates metabolic interactions between the community and the host. We also uncover the simultaneous performance of aerobic nitrification and anaerobic denitrification, which would aid to remove ammonium secreted by the sponge. Our analysis also highlights the requirement for the microbial community to respond to variable environmental conditions and hence express an array of stress protection proteins. Molecular interactions between symbionts and their host might also be mediated by a set of expressed eukaryotic-like proteins and cell-cell mediators. Finally, some sponge-associated bacteria (for example, a Phyllobacteriaceae phylotype) appear to undergo an evolutionary adaptation process to the sponge environment as evidenced by active mobile genetic elements. Our data clearly show that a combined metaproteogenomic approach can provide novel information on the activities, physiology and interactions of spongeassociated microbial communities.

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Section: Metabolismsupporting

confidence: 52%

Metaproteogenomic analysis of a community of sponge symbionts

et al. 2012

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“…FadE is the aerobic ACDH (37,38), while YdiO appears to be a member of a butyryl-CoA dehydrogenase (BCD) electron transfer protein (Etf) complex (BCD/EtfBA) necessary for growth on fatty acids under anaerobic conditions (39). Two other ACDHs could reduce crotonyl-CoA as a promiscuous substrate: the crotonobetaine reductase complex CaiAB (40,41) and AidB, the isovaleryl-CoA dehydrogenase (42,43).…”

Section: Resultsmentioning

confidence: 99%

Escherichia coli Enoyl-Acyl Carrier Protein Reductase (FabI) Supports Efficient Operation of a Functional Reversal of the β-Oxidation Cycle

Vick

Clomburg

Blankschien

et al. 2015

Appl Environ Microbiol

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We recently used a synthetic/bottom-up approach to establish the identity of the four enzymes composing an engineered functional reversal of the ␤-oxidation cycle for fuel and chemical production in Escherichia coli (J. M. Clomburg, J. E. Vick, M. D. Blankschien, M. Rodriguez-Moya, and R. Gonzalez, ACS Synth Biol 1:541-554, 2012, http://dx.doi.org/10.1021/sb3000782). While native enzymes that catalyze the first three steps of the pathway were identified, the identity of the native enzyme(s) acting as the trans-enoyl coenzyme A (CoA) reductase(s) remained unknown, limiting the amount of product that could be synthesized (e.g., 0.34 g/liter butyrate) and requiring the overexpression of a foreign enzyme (the Euglena gracilis trans-enoyl-CoA reductase [EgTER]) to achieve high titers (e.g., 3.4 g/liter butyrate). Here, we examine several native E. coli enzymes hypothesized to catalyze the reduction of enoyl-CoAs to acyl-CoAs. Our results indicate that FabI, the native enoyl-acyl carrier protein (enoyl-ACP) reductase (ENR) from type II fatty acid biosynthesis, possesses sufficient NADH-dependent TER activity to support the efficient operation of a ␤-oxidation reversal. Overexpression of FabI proved as effective as EgTER for the production of butyrate and longer-chain carboxylic acids. Given the essential nature of fabI, we investigated whether bacterial ENRs from other families were able to complement a fabI deletion without promiscuous reduction of crotonyl-CoA. These characteristics from Bacillus subtilis FabL enabled ⌬fabI complementation experiments that conclusively established that FabI encodes a native enoyl-CoA reductase activity that supports the ␤-oxidation reversal in E. coli. R ecent advances in synthetic biology and enzyme and metabolic engineering (1, 2) are allowing the development of an ever-expanding array of microbial hosts for the production of advanced fuels and industrially relevant chemicals (3, 4). Most efforts are now focused on moving past the historically successful biofuels and industrial products, such as ethanol (5), toward "drop-in" fuels and additives that can be incorporated into existing infrastructure (4). The production of such molecules requires the synthesis of higher-chain-length (C Ն 4) products from 1-, 2-or 3-carbon metabolic intermediates and hence pathways that can mediate the formation of carbon-carbon bonds. A variety of native and engineered pathways have been utilized for this purpose, including the clostridial n-butanol pathway (6), isoprenoid biosynthesis (7,8), the ␣-keto-acids pathway (9, 10), and fatty acid biosynthesis (11,12).Among the aforementioned pathways, the bacterial type II fatty acid biosynthesis system (FAB II) (13) is probably the most widely engineered and has been harnessed for the synthesis of many products, including fatty acids (14), fatty acid methyl esters (15), fatty acid ethyl esters (11, 16), fatty alcohols (11), and alkanes (12). At the core of the FAB II system is an elongation cycle that uses discrete enzymes to catalyze each of its four ste...

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“…Such a mechanism minimizes the energy costs for transport, which is particularly important as the cells grow under anaerobic conditions without a functional respiratory chain. Under anaerobic conditions, L-carnitine is reduced to ␥-butyrobetaine in a two-step pathway with crotonobetaine as intermediate (6,7,(15)(16)(17)(18). Recent studies have demonstrated that carnitine and its derivatives are activated during this process by reaction with coenzyme A (15).…”

Section: Discussionmentioning

confidence: 99%

“…The genes are organized in two divergent operons, one of which is the caiTABCDE operon. The products of the caiB and caiA genes, carnitine dehydratase and crotonobetaine reductase, have been characterized in previous studies (15)(16)(17)(18). Furthermore, the product of the caiT gene was proposed to function as a transporter based on sequence comparison and hydropathy profile analysis (13).…”

mentioning

confidence: 99%

CaiT of Escherichia coli, a New Transporter Catalyzing l-Carnitine/γ-Butyrobetaine Exchange

Jung

Buchholz

Clausen³

et al. 2002

Journal of Biological Chemistry

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L-Carnitine is essential for ␤-oxidation of fatty acids in mitochondria. Bacterial metabolic pathways are used for the production of this medically important compound. Here, we report the first detailed functional characterization of the caiT gene product, a putative transport protein whose function is required for L-carnitine conversion in Escherichia coli. The caiT gene was overexpressed in E. coli, and the gene product was purified by affinity chromatography and reconstituted into proteoliposomes. Functional analyses with intact cells and proteoliposomes demonstrated that CaiT is able to catalyze the exchange of L-carnitine for ␥-butyrobetaine, the excreted end product of L-carnitine conversion in E. coli, and related betaines. Electrochemical ion gradients did not significantly stimulate L-carnitine uptake. Analysis of L-carnitine counterflow yielded an apparent external K m of 105 M and a turnover number of 5.5 s ؊1 . Contrary to related proteins, CaiT activity was not modulated by osmotic stress. L-Carnitine binding to CaiT increased the protein fluorescence and caused a red shift in the emission maximum, an observation explained by ligand-induced conformational alterations. The fluorescence effect was specific for betaine structures, for which the distance between trimethylammonium and carboxyl groups proved to be crucial for affinity. Taken together, the results suggest that CaiT functions as an exchanger (antiporter) for L-carnitine and ␥-butyrobetaine according to the substrate/product antiport principle.

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Crotonobetaine reductase from Escherichia coli consists of two proteins

Cited by 37 publications

References 34 publications

Metaproteogenomic analysis of a community of sponge symbionts

Metaproteogenomic analysis of a community of sponge symbionts

Escherichia coli Enoyl-Acyl Carrier Protein Reductase (FabI) Supports Efficient Operation of a Functional Reversal of the β-Oxidation Cycle

CaiT of Escherichia coli, a New Transporter Catalyzing l-Carnitine/γ-Butyrobetaine Exchange

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