Volume 18, number 2 FEBS LETTERS November 1971 zyxwvutsr NON-OXIDATIVE DEMETHYLATION OF TRIMETHYLAMINE N-OXIDE BY PSEUDOMONAS AMINOVORANS P.J. LARGE Department zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA of Biochemistry, The University, Hull,HU6 7RX, England Received 10 September 197 1 1. Introduction Trimethylamine N-oxide is an important constituent of fish muscle and body fluids [l] . Most biochemical work, e.g. [2] , has concentrated on its biological reduction to trimethylamine. This paper describes the partial purification of an enzyme from methylamine- grown Pseudomonas aminovorans catalysing the non- oxidative, non-hydrolytic cleavage of trimethylamine N-oxide (equation (1)): (CH,),NO - (CH,),NH + HCHO (1) An enzyme with somewhat different properties catalysing the same reaction has recently been described in Bacillus PM6 [3]. The enzyme from l? aminovorans has a pH optimum of 6.0 and a Km for trimethylamine N-oxide of 2 mM. The equilibrium constant for reaction (1) was 6.06 mM at 25’. Triethylamine N-oxide was not a substrate, but inhibited the enzyme (Ki 8.1 mM). Other inhibitors included cyanide and mercurials. Fyridine, SKF 525-A and methylamines were not in- hibitors. The enzyme may play an important role in growth on methylamines. 2. Materials and methods 2.1. Growth and maintenance of the organism Pseudomonas aminovorans NCIB 9039 was grown as described previously [4] . 2.2. Assay for enzyme activity Trimethylamine N-oxide demethylase activity was measured in the following system: 100 poles of North-Holland fiblishing Company-Amsterdam sodium hydrogen maleate buffer pH 6.0,20 pmoles of trimethylamine N-oxide hydrochloride, enzyme and water to a final volume of 1.3 ml. The reaction was started by addition of substrate and terminated after 20 min incubation at 25” by addition of 0.2 ml of 2.5 M HClO,. After centrifuging, formaldehyde in the supernatant was estimated [5] . 2.3. Chemical estimations These were performed by the following methods: dimethylamine [6], methylamine [7] , total aliphatic aldehydes [8], protein [9] , and trimethylamine N-oxide [lo]. When enzymically-formed formaldehyde was present in the same solution as trimethylamine N-oxide, it was first eliminated by oxidation to formate as follows. The perchloric acid was neutralised by addition of 0.1 of a volume of 2.5 M NaOH. Then 0.05 ml of 100 volume H,O, was added and the samples incubated at 50” for 10 min. The trimethylamine N-oxide was then estimated by the above method. 2.4. Purification of the enzyme The enzyme was unstable when purified more than about 5-fold, and higher purifications were not ob- tained. The following method gave a reasonably stable preparation. Crude ultrasonic extracts, after centrifuging at 25,000 g for 20 min, were made 50% saturated by addition of solid ammonium sulphate. The precipit- ated protein was removed by centrifuging at 25,000 g for 20 min at 0” and redissolved in 10 mM phosphate pH 7.4 and dialysed at 4” against the same buffer for 4 hr. It was then applied to a column (30 cm X 2 cm) of DEAEcellulose (Whatman DE32) and eluted at 4” 297