title: Tropine dehydrogenase: purification, some properties and an evaluation of its role in the bacterial metabolism of tropine.
abstract: Tropine dehydrogenase was induced by growth of Pseudomonas AT3 on atropine, tropine or tropinone. It was NADP(+)-dependent and gave no activity with NAD+. The enzyme was very unstable but a rapid purification procedure using affinity chromatography that gave highly purified enzyme was developed. The enzyme gave a single band on isoelectric focusing with an isoelectric point at approximately pH 4. The native enzyme had an M(r) of 58,000 by gel filtration and 28,000 by SDS/PAGE and therefore consists of two subunits of equal size. The enzyme displayed a narrow range of specificity and was active with tropine and nortropine but not with pseudotropine, pseudonortropine, or a number of related compounds. The apparent Kms were 6.06 microM for tropine and 73.4 microM for nortropine with the specificity constant (Vmax/Km) for tropine 7.8 times that for pseudotropine. The apparent Km for NADP+ was 48 microM. The deuterium of [3-2H]tropine and [3-2H]pseudotropine was retained when these compounds were converted into 6-hydroxycyclohepta-1,4-dione, an intermediate in tropine catabolism, showing that the tropine dehydrogenase, although induced by growth on tropine, is not involved in the catabolic pathway for this compound. 6-Hydroxycyclohepta-1,4-dione was also implicated as an intermediate in the pathways for pseudotropine and tropinone catabolism.
authors.affiliation: Institute of Biological Sciences, University of Wales, Aberystwyth, Dyfed, U.K.
authors.forename: B A
authors.initials: BA
authors.lastname: Bartholomew
authors.forename: M J
authors.initials: MJ
authors.lastname: Smith
authors.forename: M T
authors.initials: MT
authors.lastname: Long
authors.forename: P J
authors.initials: PJ
authors.lastname: Darcy
authors.forename: P W
authors.initials: PW
authors.lastname: Trudgill
authors.forename: D J
authors.initials: DJ
authors.lastname: Hopper
mesh_terms: D000429:Alcohol Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D008401:Gas Chromatography-Mass Spectrometry; D009249:NADP / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D013379:Substrate Specificity; D014326:Tropanes / Q000378:metabolism*
chemical_list: D014326:Tropanes; D009249:NADP; C005864:tropine; D000429:Alcohol Oxidoreductases; C091007:tropine dehydrogenase