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

Biochemistry. 2001 Sep 18;40(37):11140-8. doi: 10.1021/bi0108812.

Abstract

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).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyl Coenzyme A / metabolism*
  • Acyltransferases / immunology
  • Acyltransferases / metabolism*
  • Amino Acid Sequence
  • Antibodies, Monoclonal
  • Betaine / analogs & derivatives*
  • Betaine / metabolism*
  • Carnitine / biosynthesis*
  • Enoyl-CoA Hydratase / immunology
  • Enoyl-CoA Hydratase / metabolism*
  • Escherichia coli / enzymology*
  • Escherichia coli Proteins*
  • Models, Chemical
  • Molecular Sequence Data
  • Proteus / enzymology
  • Racemases and Epimerases / immunology
  • Racemases and Epimerases / metabolism
  • Sequence Homology, Amino Acid

Substances

  • Acyl Coenzyme A
  • Antibodies, Monoclonal
  • Escherichia coli Proteins
  • crotonobetainyl-CoA
  • Betaine
  • gamma-butyrobetaine
  • crotonic acid betaine
  • Acyltransferases
  • crotonobetainyl-CoA carnitine coenzyme A transferase
  • Enoyl-CoA Hydratase
  • Racemases and Epimerases
  • caiD protein, E coli
  • Carnitine