Purification and properties of carnitine dehydratase from Escherichia coli — a new enzyme of carnitine metabolization

Jung, Heinrich; Jung, Kirsten; Kleber, Hans‐Peter

doi:10.1016/0005-2760(89)90232-4

Cited by 55 publications

(84 citation statements)

References 23 publications

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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).…”

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confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

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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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“…The nucleotide sequence of CDHT is shown in Fig. 3 (8) for the purified CDHT. In addition, the previously determined N-terminal sequence of purified CDHT was compared with the deduced amino acid sequence from the sequenced ORF.…”

Section: Methodsmentioning

confidence: 99%

“…L-( -)-CDHT was purified and characterized by its kinetic and molecular properties (7,8). During the purification process, a thermostable, low-molecular-weight factor which is essential for enzyme activity was lost.…”

Section: L-( -)-Carnitine [R-( -)-3-hydroxy-4-trimethylaminobutyrmentioning

confidence: 99%

Cloning, nucleotide sequence, and expression of the Escherichia coli gene encoding carnitine dehydratase

Eichler¹,

Schunck²,

Kleber³

et al. 1994

J Bacteriol

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Carnitine dehydratase from Escherichia coli O44 K74 is an inducible enzyme detectable in cells grown anaerobically in the presence of L-(-)-carnitine or crotonobetaine. The purified enzyme catalyzes the dehydration of L-(-)-carnitine to crotonobetaine (H. Jung, K. Jung, and H.-P. Kleber, Biochim. Biophys. Acta 1003:270-276, 1989). The caiB gene, encoding carnitine dehydratase, was isolated by oligonucleotide screening from a genomic library of E. coli O44 K74. The caiB gene is 1,215 bp long, and it encodes a protein of 405 amino acids with a predicted M(r) of 45,074. The identity of the gene product was first assessed by its comigration in sodium dodecyl sulfate-polyacrylamide gels with the purified enzyme after overexpression in the pT7 system and by its enzymatic activity. Moreover, the N-terminal amino acid sequence of the purified protein was found to be identical to that predicted from the gene sequence. Northern (RNA) analysis showed that caiB is likely to be cotranscribed with at least one other gene. This other gene could be the gene encoding a 47-kDa protein, which was overexpressed upstream of caiB.

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“…This enzyme catalyzes the dehydration of carnitine [R(Ϫ)3-hydroxy-4-trimethylaminobutyrate] to crotonobetaine. There is also no free amino group present in carnitine; therefore, pyridoxal phosphate is not the coenzyme of the reaction (19). The cofactor required for carnitine dehydratase is not known; whether it is an iron-sulfur cluster or a new cofactor has not been investigated.…”

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Expression, purification, and characterization of EpiC, an enzyme involved in the biosynthesis of the lantibiotic epidermin, and sequence analysis of Staphylococcus epidermidis epiC mutants

Kupke

Götz

1996

J Bacteriol

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Purification and properties of carnitine dehydratase from Escherichia coli — a new enzyme of carnitine metabolization

Cited by 55 publications

References 23 publications

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

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

Cloning, nucleotide sequence, and expression of the Escherichia coli gene encoding carnitine dehydratase

Expression, purification, and characterization of EpiC, an enzyme involved in the biosynthesis of the lantibiotic epidermin, and sequence analysis of Staphylococcus epidermidis epiC mutants

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