Two proteins (CaiB and CaiD) were found to catalyze the reversible biotransformation of crotonobetaine to L-carnitine in Escherichia coli in the presence of a cosubstrate (e.g., gamma-butyrobetainyl-CoA or crotonobetainyl-CoA). CaiB (45 kDa) and CaiD (27 kDa) were purified in two steps to electrophoretic homogeneity from overexpression strains. CaiB was identified as crotonobetainyl-CoA:carnitine CoA-transferase by MALDI-TOF mass spectrometry and enzymatic assays. The enzyme exhibits high cosubstrate specificity to CoA derivatives of trimethylammonium compounds. In particular, the N-terminus of CaiB shows significant identity with other CoA-transferases (e.g., FldA from Clostridium sporogenes, Frc from Oxalobacter formigenes, and BbsE from Thauera aromatica) and CoA-hydrolases (e.g., BaiF from Eubacterium sp.). CaiD was shown to be a crotonobetainyl-CoA hydratase using MALDI-TOF mass spectrometry and enzymatic assays. Besides crotonobetainyl-CoA CaiD is also able to hydrate crotonyl-CoA with a significantly lower Vmax (factor of 10(3)) but not crotonobetaine. The substrate specificity of CaiD and its homology to the crotonase confirm this enzyme as a new member of the crotonase superfamily. Concluding these results, it was verified that hydration of crotonobetaine to L-carnitine proceeds at the CoA level in two steps: the CaiD catalyzed hydration of crotonobetainyl-CoA to L-carnitinyl-CoA, followed by a CoA transfer from L-carnitinyl-CoA to crotonobetaine, catalyzed by CaiB. When gamma-butyrobetainyl-CoA was used as a cosubstrate (CoA donor), the first reaction is the CoA transfer. The optimal ratios of CaiB and CaiD during this hydration reaction, determined to be 4:1 when crotonobetainyl-CoA was used as cosubstrate and 5:1 when gamma-butyrobetainyl-CoA was used as cosubstrate, are different from that found for in vivo conditions (1:3).
A still unknown low-molecular-mass cofactor essential for the activity of carnitine-metabolizing enzymes (e.g., L-carnitine dehydratase, crotonobetaine reductase) from E. coli has been purified to homogeneity from a cell-free extract of E. coli O44K74. The purity of the cofactor was confirmed by HPLC analysis. Biosynthesis of the unknown compound was only observed when bacteria were cultivated anaerobically in the presence of L-carnitine or crotonobetaine. The determined properties, together with results obtained from UV-visible, (1)H NMR, and mass spectrometry, indicate that the compound in question is a new CoA derivative. The esterified compound was suggested to be gamma-butyrobetaine-a metabolite of carnitine metabolism of E. coli. Proof of structure was performed by chemical synthesis. Besides gamma-butyrobetainyl-CoA, a second new CoA derivative, crotonobetainyl-CoA, was also chemically synthesized. Both CoA derivatives were purified and their structures confirmed using NMR and mass spectrometry. Comparisons of structural data and of the chemical properties of gamma-butyrobetainyl-CoA, crotonobetainyl-CoA, and the isolated cofactor verified that the unknown compound is gamma-butyrobetainyl-CoA. The physical and chemical properties of gamma-butyrobetainyl-CoA and crotonobetainyl-CoA are similar to known CoA derivatives.
The focus of our work is the identification of medically relevant staphylococci from the environment; these organisms are among the major opportunistic pathogens associated with human disease. Andersen sampling was performed in schoolrooms during the school year. Eleven of thirty six isolates (all Gram-positive tetrads) were identified as staphylococci and 23 were characterized as micrococci. MALDI-TOF MS profiling was used as the first stage in the classification followed by standard biochemical tests including API Staph profiling. The staphylococcal isolates were each speciated; coagulase positive (Staphylococcus aureus [3 strains]) and coagulase negative: Staphylococcus warneri (4 isolates), Staphylococcus hominis (2), Staphylococcus saprophyticus (1) and Staphylococcus cohnii (1). S. aureus is most commonly found in the human nares but is frequently isolated from skin. The other staphylococcal species are among those most commonly isolated from human skin. Micrococci were much more frequently isolated from indoor air than reported by others for clinical samples. It is suggested that, without discrimination from micrococci, misidentification of staphylococci would be common on air sampling.
Enzymes involved in carnitine metabolism of Proteus sp. are encoded by the cai genes organised as the caiTABCDEF operon. The complete operon could be sequenced from the genomic DNA of Proteus sp. Amino acid sequence similarities and/or enzymatic analysis confirmed the function assigned to each protein involved in carnitine metabolism. CaiT was suggested to be an integral membrane protein responsible for the transport of betaines. The caiA gene product was shown to be a crotonobetainyl-CoA reductase catalysing the irreversible reduction of crotonobetainyl-CoA to gamma-butyrobetainyl-CoA. CaiB and CaiD were identified to be the two components of the crotonobetaine hydrating system, already described. CaiB and caiD were cloned and expressed in Escherichia coli. After purification of both proteins, their individual enzymatic functions were solved. CaiB acts as betainyl-CoA transferase specific for carnitine, crotonobetaine, gamma-butyrobetaine and its CoA derivatives. Transferase reaction proceeds, following a sequential bisubstrate mechanism. CaiD was identified to be a crotonobetainyl-CoA hydratase belonging to the crotononase superfamily. Because of amino acid sequence similarities, CaiC was suggested to be a betainyl-CoA ligase. Taken together, these results show that the metabolism of carnitine and crotonobetaine in Proteus sp. proceeds at the CoA level.
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