Purification and characterization of d(+)-carnitine dehydrogenase from Agrobacterium sp. — a new enzyme of carnitine metabolism

Abstract d (+)-Carnitine dehydrogenase from Agrobacterium sp. catalyzes the oxidation of d (+)-carnitine to 3-dehydrocarnitine as initial step of d (+)-carnitine degradation. The NAD + -specific, cytosolic enzyme was purified 126-fold to apparent electrophoretic homogeneity by 4 chromatographic steps. The molecular mass of the native enzyme was estimated to be 88 kDa by size-exclusion chromatography. It seems to be composed of 3 identical subunits with a relative molecular mass of 28 kDa as found by sodium dodecyl sulfate polyacrylamide gel electrophoresis and laser-induced mass spectrometry. The isoelectric point was found to be 4.7–5.0. The optimum temperature is 37°C and the optimum pH for the oxidation and the reduction reaction are 9.0–9.5 and 5.5–6.5, respectively. The purified enzyme was further characterized with respect to substrate specificity, kinetic parameters and amino terminal sequence. Analogues of d (+)-carnitine ( l (−)-carnitine, crotonobetaine, γ-butyrobetaine, carnitine amide, glycine betaine, choline) are competitive inhibitors of d (+)-carnitine oxidation. The equilibrium constant of the reaction of d (+)-carnitine dehydrogenase was determined to be 2.2 × 10 −12 . The purified d (+)-carnitine dehydrogenase has similar kinetic properties to the l (−)-carnitine dehydrogenase from the same microorganism as well as to l (−)-carnitine dehydrogenases of other bacteria.