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

Abstract

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.

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

“…Cells containing plasmid pT7-5 without caiT showed no or only little uptake of L-[methyl- 14 (Fig. 2).…”

“…L-[methyl- 14 C]Carnitine uptake was measured at substrate concentrations from 2 to 50 M at 25°C. For analysis of the Na ϩ dependence of transport, 50 mM NaCl was added immediately prior to addition of L-[methyl-…”

“…The resulting cell suspension was diluted 200-fold into resuspension buffer containing L-[methyl- 14 C]carnitine (53 Ci/mol) at a concentration of 4.7 M. In the case of counterflow experiments (cells preloaded with 10 mM L-carnitine), this yielded a final external L-carnitine concentration of 54.7 M. Incubation was carried out at 25°C, and transport was terminated at different time points by addition of 100 mM potassium phosphate buffer (pH 6.0) and 100 mM LiCl (stop buffer) and rapid filtration as described (29).…”

“…Ion-dependent transport was started by 200-fold dilution of aliquots of the proteoliposome suspension (2 l) into buffer containing 100 mM Tris/Mes (pH 6.0), 2 mM ␤-mercaptoethanol, 5 mM MgSO 4 , and 0.2 M valinomycin (creation of ⌬ H ϩ) or 100 mM Tris/Mes (pH 7.5), 2 mM ␤-mercaptoethanol, 5 mM MgSO 4 , 0.2 M valinomycin, and 50 mM NaCl (creation of ⌬ Na ϩ). The buffers contained L-[methyl- 14 C]carnitine at concentrations varying between 2 and 50 M.…”

“…The E. coli genes responsible for carnitine conversion have been cloned and sequenced (12)(13)(14). The genes are organized in two divergent operons, one of which is the caiTABCDE operon.…”