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The biotransformation of crotonobetaine and D(+)-carnitine into L(-)-carnitine is affected by salt stress in the resting cells of E. coli O44 K74 and the transformed E. coli K38 pT7-5KE32. A yield of 65 and 80% of L(-)-carnitine, respectively, were obtained with 0.5 M NaCl with the wild and transformed strain compared with the 40% obtained with the control. Higher salt levels reduced the conversion. In L(-)-carnitine transport studies using both strains, the transformed strain presented slightly lower apparent K(m) and V values. Arsenate reduced both the transport and biotransformation of crotono-betaine in the presence or absence of 0.5 M NaCl, whereas vanadate only inhibited these processes under salt stress conditions. Hg(II) inhibited both the transport and biotransformation and Pb(II) reduced the biotransformation only under salt stress conditions. Cu(II) produced a significantly higher decrease than Pb(II) in the biotransformation with both substrates in the absence of salt stress conditions, but only affected transport in the presence of such conditions. Furthermore, salt stress affected the CaiT transporter for L(-)-carnitine and crotonobetaine and induced ProU and ProP in the absence of the inducer of the L(-)-carnitine metabolism. It is highly likely that the increase in L(-)-carnitine production was not only due to improved transport but also to the permeabilization effect caused by NaCl, as transport and 1-N-phenylnaphthylamine uptake studies revealed.

Two proteins, component I (CI) and component II (CII), catalyze the biotransformation of crotonobetaine to L(–)-carnitine in Proteus sp. CI was purified to electrophoretic homogeneity from cell-free extracts of Proteus sp. The N-terminal amino acid sequence of CI showed high similarity (80%) to the caiB gene product from Escherichia coli O44K74, which encodes the L(–)-carnitine dehydratase. CI alone was unable to convert crotonobetaine into L(–)-carnitine even in the presence of the cosubstrates crotonobetainyl-CoA or γ-butyrobetainyl-CoA, which are essential for this biotransformation. The relative molecular mass of CI was determined to be 91.1 kDa. CI is composed of two identical subunits of molecular mass 43.6 kDa. The isoelectric point is 5.0. CII was purified to electrophoretic homogeneity from cell-free extracts of Proteus sp. and its N-terminal amino acid sequence showed high similarity (75%) to the caiD gene product of E. coli O44K74. The relative molecular mass of CII was shown to be 88.0 kDa, and CII is composed of three identical subunits of molecular mass 30.1 kDa. The isoelectric point of CII is 4.9. For the biotransformation of crotonobetaine to L(–)-carnitine, the presence of CI, CII, and a cosubstrate (crotonobetainyl-CoA or γ-butyrobetainyl-CoA) were shown to be essential. [ABSTRACT FROM AUTHOR]

The sequence encompassing the cai genes of Escherichia coli, which encode the carnitine pathway, has been determined. Apart from the already identified caiB gene coding for the carnitine dehydratase, five additional open reading frames were identified. They belong to the caiTABCDE operon, which was shown to be located at the first minute on the chromosome and transcribed during anaerobic growth in the presence of carnitine. The activity of carnitine dehydratase was dependent on the CRP regulatory protein and strongly enhanced in the absence of a functional H-NS protein, in relation to the consensus sequences detected in the promoter region of the cai operon. In vivo expression studies led to the synthesis of five polypeptides in addition to CaiB, with predicted molecular masses of 56 613 Da (CaiT), 42 564 Da (CaiA), 59311 Da (CaiC), 32 329 Da (CaiD) and 21 930 Da (CaiE). Amino acid sequence similarity or enzymatic analysis supported the function assigned to each protein. CaiT was suggested to be the transport system for carnitine or betaines, CaiA an oxidoreduction enzyme, and CaiC a crotonobetaine/carnitine CoA ligase. CaiD bears strong homology with enoyl hydratases/isomerases. Overproduction of CaiE was shown to stimulate the carnitine racemase activity of the CaiD protein and to markedly increase the basal level of carnitine dehydratase activity. It is inferred that CaiE is an enzyme involved in the synthesis or the activation of the still unknown cofactor required for carnitine dehydratase and carnitine racemase activities. Taken together, these data suggest that the carnitine pathway in E. coli resembles that found in a strain situated between Agrobacterium and Rhizobium. [ABSTRACT FROM AUTHOR]

Investigates the biotransformation of crotonobetaine to L-carnitine in Escherichia coli. Hydration of crotonobetaine to L-carnitine; Role of coenzyme A esters, enoyl-CoA hydratase and CoA-transferase in the biotransformation; Effect of cosubstrate on the hydration reaction; Reversibility of crotonobetaine bioconversion.