Involvement of coenzyme A esters and two new enzymes, an enoyl-CoA hydratase and a CoA-transferase, in the hydration of crotonobetaine to L-carnitineby Escherichia coli

Two proteins (CaiB and CaiD) were found to catalyze the reversible biotrans formation of crotonobetaine to L-carnitine in Escherichia coli in the prese nce of a cosubstrate (e.g., gamma -butyrobetainyl-CoA or crotonobetainyl-Co A). CaiB (45 kDa) and CaiD (27 kDa) were purified in two steps to electroph oretic homogeneity from overexpression strains. CaiB was identified as crot onobetainyl-CoA: carnitine CoA-transferase by MALDI-TOF mass spectrometry a nd enzymatic assays. The enzyme exhibits high cosubstrate specificity to Co A 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 fro m Thauera aromatica) and CoA-hydrolases (e.g., BaiF from Eubacterium sp.). CaiD was shown to be a crotonobetainyl-CoA hydratase using MALDI-TOF mass s pectrometry and enzymatic assays. Besides crotonobetainyl-CoA CaiD is also able to hydrate crotonyl-CoA with a significantly lower V-max (factor of 10 (3)) but not crotonobetaine. The substrate specificity of CaiD and its homo logy to the crotonase confirm this enzyme as a new member of the crotonase superfamily. Concluding these results, it was verified that hydration of cr otonobetaine to L-carnitine proceeds at the CoA level in two steps: the Cai D catalyzed hydration of crotonobetainyl-CoA to L-carnitinyl-CoA, followed by a CoA transfer from L-carnitinyl-CoA to crotonobetaine, catalyzed by Cai B. When gamma -butyrobetainyl-CoA was used as a cosubstrate (CoA donor), th e first reaction is the CoA transfer. The optimal ratios of CaiB and CaiD d uring this hydration reaction, determined to be 4:1 when crotonobetainyl-Co A was used as cosubstrate and 5:1 when gamma -butyrobetainyl-CoA was used a s cosubstrate, are different from that found for in vivo conditions (1:3).