A soluble y-butyrobetaine hydroxylase has been partially purified from cells of a Pseudomonas strain (Pseudomonas sp AK 1) by chromatography on DEAE-cellulose and on hydroxylapatite. Carnitine is the only product of y-butyrobetaine; trimethylamine or trimethylaminoacetone is not formed. The enzyme has a low isoelectric point (around pH 4.5). The K M ,~~~ value for y-butyrobetaine is 2.4 mM. There is an absolute and specific requirement for 2-ketoglutarate and I n the previous studies of carnitine metabolism, evidence was obtained for a low turnover rate of carnitine in the rat (Lindstedt and Lindstedt, 1961a). Of various hypothetical precursors, only y-butyrobetaine (4-trimethylaminobutyrate) was converted efficiently into carnitine Lindstedt, 1961 b, 1965a). Results from studies with preparations from rat liver indicated an oxygenase type of mechanism for this reaction (Lindstedt and Lindstedt, 1962;. The pathway for carnitine degradation in the rat is unknown. A poxidation type of reaction for the degradation of carnitine in Pseudomonas aeruginosa NCTC A 7244 was suggested from the finding of glycine betaine as a metabolite of carnitine (Lindstedt and Lindstedt, 1962). The possibility that this type of mechanism occurred in the formation of carnitine from ybutyrobetaine in bacteria was therefore considered. A Pseudomonas strain (Pseudomonas sp AK 1) was obtained by enrichment culture on y-butyrobetaine media (Lindstedt et af., 1967b). Carnitine was formed from y-butyrobetaine in incubations with acetone-dried cells under conditions which suggested a monooxygenase reaction. The hydroxylase from Pseudomonas sp AK 1 has been used for studies of the reaction mechanism (Lindblad et al., 1969) as high specific activity is more easily obtained with the bacterial preparations than with those from rat liver. The partial purification of the enzyme and studies of cofactor requirements are now presented. Some of the results have been reported in preliminary form (Lindstedt et af., 1967a,c).
Material and MethodsChemicals. Chemicals were obtained from the following sources: y-butyrobetaine chloride, ferrous sulfate, and 1,lOphenanthroline from E. Merck, AG, Darmstadt, West Germany; oxalacetic acid, mercaptoethanol, L-glutamic acid, sodium DL-isocitrate, zinc 2-hydroxyglutarate, sodium pyruvate, succinic acid, isoascorbate, dichlorophenolindophenol, and
