Molecular and biochemical characterization of rat γ- trimethylaminobutyraldehyde dehydrogenase and evidence for the involvement of human aldehyde dehydrogenase 9 in carnitine biosynthesis

Abstract

The penultimate step in carnitine biosynthesis is mediated by γ- trimethylaminobutyraldehyde dehydrogenase (EC 1.2.1.47), a cytosolic NAD+- dependent aldehyde dehydrogenase that converts γ-trimethylaminobutyraldehyde into γ-butyrobetaine. This enzyme was purified from rat liver, and two internal peptide fragments were sequenced by Edman degradation. The peptide sequences were used to search the Expressed Sequence Tag data base, which led to the identification of a rat cDNA containing an open reading frame of 1485 base pairs encoding a polypeptide of 494 amino acids with a calculated molecular mass of 55 kDa. Expression of the coding sequence in Escherichia coli confirmed that the cDNA encodes γ-trimethylaminobutyraldehyde dehydrogenase. The previously identified human aldehyde dehydrogenase 9 (EC 1.2.1.19) has 92% identity with rat trimethylaminobutyraldehyde dehydrogenase and has been reported to convert substrates that resemble γ- trimethylaminobutyraldehyde. When aldehyde dehydrogenase 9 was expressed in E. coli, it exhibited high trimethylaminobutyraldehyde dehydrogenase activity. Furthermore, comparison of the enzymatic characteristics of the heterologously expressed human and rat dehydrogenases with those of purified rat liver trimethylaminobutyraldehyde dehydrogenase revealed that the three enzymes have highly similar substrate specificities. In addition, the highest V(max)/K(m) values were obtained with γ-trimethylaminobutyraldehyde as substrate. This indicates that human aldehyde dehydrogenase 9 is the γ- trimethylaminobutyraldehyde dehydrogenase, which functions in carnitine biosynthesis.