Purification and characterization of D(+)-carnitine dehydrogenase from Agrobacterium sp.--a new enzyme of carnitine metabolism.
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 degrees 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, gamma-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 x 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.
Hanschmann
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Institut für Biochemie, Fakultät für Biowissenschaften, Pharmazie und Psychologie, Universität Leipzig, Germany.
Kleber
H P
HP
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002331:Carnitine / Q000031:analogs & derivatives / Q000378:metabolism; D002845:Chromatography / Q000379:methods; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors; D007526:Isoelectric Point; D007536:Isomerism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D012231:Rhizobium / Q000201:enzymology*; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D009243:NAD; C103930:3-dehydrocarnitine; D000429:Alcohol Oxidoreductases; C019842:carnitine dehydrogenase; D002331:Carnitine