Involvement of coenzyme A esters and two new enzymes, an enoyl-CoA hydratase and a CoA-transferase, in the hydration of crotonobetaine to L-carnitine by Escherichia coli.
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).
Elssner
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T
Institut für Biochemie, Fakultät für Biowissenschaften, Pharmazie, und Psychologie, Universität Leipzig, Talstrasse 33, D-04103 Leipzig, Germany. elssner@uni-leipzig.de
Engemann
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C
Baumgart
K
K
Kleber
H P
HP
D000214:Acyl Coenzyme A / Q000378:metabolism*; D000217:Acyltransferases / Q000276:immunology / Q000378:metabolism*; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001622:Betaine / Q000031:analogs & derivatives* / Q000378:metabolism*; D002331:Carnitine / Q000096:biosynthesis*; D004746:Enoyl-CoA Hydratase / Q000276:immunology / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011511:Proteus / Q000201:enzymology; D019998:Racemases and Epimerases / Q000276:immunology / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D000214:Acyl Coenzyme A; D000911:Antibodies, Monoclonal; D029968:Escherichia coli Proteins; C413607:crotonobetainyl-CoA; D001622:Betaine; C002889:gamma-butyrobetaine; C025237:crotonic acid betaine; D000217:Acyltransferases; C060278:crotonobetainyl-CoA carnitine coenzyme A transferase; D004746:Enoyl-CoA Hydratase; D019998:Racemases and Epimerases; C091404:caiD protein, E coli; D002331:Carnitine