Tropine dehydrogenase: purification, some properties and an evaluation of its role in the bacterial metabolism of tropine.
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.
Bartholomew
B A
BA
Institute of Biological Sciences, University of Wales, Aberystwyth, Dyfed, U.K.
Smith
M J
MJ
Long
M T
MT
Darcy
P J
PJ
Trudgill
P W
PW
Hopper
D J
DJ
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*
D014326:Tropanes; D009249:NADP; C005864:tropine; D000429:Alcohol Oxidoreductases; C091007:tropine dehydrogenase