By techniques involving differential centrifugation and specific precipitation with CaCI, it was shown that dimethylamine and trimethylamine mono-oxygenase activities co-sediment with NADPH-cytochrome c reductase activity in sphaeroplast lysates of Candida utilis grown on trimethylamine as sole nitrogen source. Since the active fraction also contained low levels of cytochromes P-450 and P-420, it was concluded that the two amine mono-oxygenases are located in the smooth endoplasmic reticulum and thus end up in the microsomal fraction on cell fractionation. Ten to twenty-fold enrichment of mono-oxygenase specific activity could be achieved by separation of activity from soluble protein by centrifugation or gel filtration. Cell-free extracts prepared in the absence of FAD showed only very low mono-oxygenase activity for either substrate. Some activity could be restored by addition of flavin nucleotides: there was a fivefold stimulation by FAD and a fourfold stimulation by FMN. All trimethylamine mono-oxygenase activity was lost when a partially purified preparation containing both activities was incubated for more than 24 h at 0°C, suggesting that separate enzymes are responsible for the oxidation of secondary and tertiary amines. The enzyme preparation oxidized a wide range of secondary alkylamines up to dibutylamine and tertiary alkylamines up to tributylamine. Primary amines, choline, di-and triethanolamine, spermine, spermidine and substituted anilines were not oxidized. NADH had a lower apparent Km value and higher Vmax value than NADPH. Secondary and tertiary alkylamines containing more than one kind of alkyl group gave more than one kind of aldehyde on oxidation. Stoicheiometry determinations showed a consumption of 1 mol NAD(P)H and 1 mol O2 per mol aldehyde formed. Carbon monoxide, cyanide, proadifen hydrochloride (SKF 525-A), mercurials and mercaploethanol all inhibited both activities.
BartelsP.D., JensenP.K.1979; Role of AMP in regulation of the citric acid cycle in mitochondria from bakers’ yeast. Biochimica et biophysica acta 582:246–259
BoultonC.A., CrabbeM.J.C., LargeP.J.1974; Microbial oxidation of amines. Partial purification of a trimethylamine monooxygenase from Pseudomonas aminovorans and its role in growth on trimethylamine. Biochemical Journal 140:253–263
BradfordM.M.1976; A rapid and sensitive method for the quantitative determination of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248–254
BrookD.F., LargeP.J.1975; Inhibition by carbon monoxide of the secondary-amine monooxygenase of Pseudomonas aminovorans and the photochemical action spectrum for its reversal. European Journal of Biochemistry 55:601–609
DelaisséJ.-M., MartinP., Verheyen-BouvyM.-F., NyssE.J.1981; Subcellular distribution of enzymes in the yeast Saccharomycopsis lipolytica, grown on n-hexadecane, with special reference to the ω-hydroxylase. Biochimica et biophysica acta 676:77–90
EadyR.R., JarmanR.T., LargeP.J.1971; Microbial oxidation of amines. Partial purification of a mixed-function secondary-amine oxidase system from Pseudomonas aminovorans that contains an enzymically active cytochrome P-420-type haemoprotein. Biochemical Journal 125:449–459
GreenJ., LargeP.J.1983a; Oxidation of dimethylamine and trimethylamine in methazotrophic yeasts by microsomal mono-oxygenases sensitive to carbon monoxide. Biochemical and Biophysical Research Communications 113:900–907
GreenJ., LargeP.J.1983b; Cell-free oxidation of dimethylamine by a mono-oxygenase in the methazotrophic yeast Candida utilis. Biochemical Society Transactions 11:786
GreenJ., LargeP.J.1984; Regulation of the key enzymes of methylated amine metabolism in Candida boidinii. Journal of General Microbiology 130:1947–1959
HaywoodG.W., LargeP.J.1981; Microbial oxidation of amines. Distribution, purification and properties of two primary-amine oxidases from the yeast Candida boidinii grown on amines as sole nitrogen source. Biochemical Journal 199:187–201
HlavicaP.1982; Biological oxidation of nitrogen in organic compounds and disposition of N-oxidized products. CRC Critical Reviews of Biochemistry 12:39–101
JenkinsR.O., CartledgeT.G., LloydD.1983; Subcellular fractionation of Candida stellatoidea aftergrowth with glucose or n-hexadecane. Journal of General Microbiology 129:1171–1185
LargeP.J.1981; Microbial growth on methylated amines. In Microbial Growth on C1 Compounds. Proceedings of Third International Symposium pp. 55–69DaltonH. Edited by London: Heyden & Son;
LargeP.J., GreenJ.1984; Oxidation of mono-, di-and trimethylamine by methazotrophic yeasts: properties of the microsomal and peroxisomal enzymes involved and comparison with bacterial systems. In Microbial Growth on C1 Compounds. Proceedings of Fourth International Symposium.CrawfordR.L. Edited by Washington: American Society for Microbiology; (in the Press)
LargeP.J., EadyR.R., MurdenD.J.1969; An enzymic method for the microestimation of methyl-amine, ethylamine, and n-propylamine. Analytical Biochemistry 32:402–407
MeibergJ.B.M., HarderW.1978; Aerobic and anaerobic metabolism of trimethylamine, dimethylamine and methylamine in Hyphomicrobium X. Journal of General Microbiology 106:265–276
OsumiM., MiwaN., TeranishiY., TanakaA., FukuiS.1975; Development of microbodies in Candida tropicalis during incubation in an-alkane medium. Archives of Microbiology 103:1–11
PettitF.H., Orme-JohnsonW., ZieglerD.M.1964; The requirement for flavin adenine dinucleotide by a liver microsomal oxygenase catalyzing the oxidation of alkylaryl amines. Biochemical and Biophysical Research Communications 16:444–448
RobinsonJ., CooperJ.M.1970; Method of determining oxygen concentrations in biological media, suitable for calibration of the oxygen electrode. Analytical Biochemistry 33:390–399
SawickiE., HauserT.R., StanleyT.W., ElbertW.1961; The 3-methyl-2-benzothiazolone hydrazone test. Sensitive new methods for the detection, rapid estimation and determination of aliphatic aldehydes. Analytical Chemistry 33:93–96
ShaltielS., Er-ELZ.1973; Hydrophobic chromatography: use for purification of glycogen synthetase. Proceedings of the National Academy of Sciences of the United States of America 70:778–781
TanakaA., YasuharaS., KawamotoS., FukuiS., OsumiM.1976; Development of microbodies in the yeast Kloeckera growing on methanol. Journal of Bacteriology 126:919–927
VeenhuisM., Van DijkenJ.P., HarderW.1980; A new method for the cytochemical demonstration of phosphatase activities in yeasts based on the use of cerous ions. FEMS Microbiology Letters 9:285–291
VeenhuisM., Van DijkenJ.P., HarderW.1983; The significance of peroxisomes in the metabolism of one-carbon compounds in yeasts. Advances in Microbial Physiology 24:1–82
YamadaT., TanakaA., FukuiS.1982; Properties of catalase purified from whole cells and peroxisomes of n-alkane-grown Candida tropicalis. European Journal of Biochemistry 125:517–521
ZatmanL.J.1981; A search for patterns in methylotrophic pathways.In Microbial Growth on C1Compounds. Proceedings of Third International Symposium pp. 42–54DaltonH. Edited by London: Hcydcn & Son;
ZwartK.B., HarderW.1983; Regulation of the metabolism of some alkylated amines in the yeasts Candida utilis and Hansenula polymorpha. Journal of General Microbiology 129:3157–3169
ZwartK., VeenhuisM., Van DijkenJ.P., HarderW.1980; Development of amine-oxidase-containing peroxisomes in yeasts during growth on glucose in the presence of mcthylamine as the sole source of nitrogen. Archives of Microbiology 126:117–126
ZwartK.B., VeenhuisM., PlatG., HarderW.1983; Characterization of glyoxysomes in yeast and their transformation into peroxisomes in response to changes in environmental conditions. Archives of Microbiology 136:28–38