Cloning and Characterization of Isopropanol Dehydrogenase from Phytomonas Sp.

MOLINAS, SARA M.; ALTABE, SILVIA; RIDER, MARK; OPPERDOES, FRED; UTTARO, ANTONIO

Carlos Paz. Córdoba. Argentina

Congreso; XXXVII Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular; 2001

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular

CLONING AND CHARACTERIZATION OF ISOPROPANOL DEHYDROGENASE FROM PHYTOMONAS SP. Molinas, Sara#; Altabe, Silvia#; Rider, Mark**; Opperdoes, Fred*; Uttaro, Antonio#   #IBR-CONICET-Dto de Microbiología. Facultad de Cs. Bioquímicas y Farmacéuticas. Universidad Nacional de Rosario. ** Research Unit for Hormone and Metabolism. * Research Unit for Tropical Diseases. IC   XXXVII Reunión Anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular. Del 20 al 23 de Noviembre del 2001. Carlos Paz. Argentina    We have described previously the carbohydrate metabolism of Phytomonas sp. Isolated from latex-bearing spurge Euphorbia characias. It showed to be very similar to the metabolism of bloodstream form of Trypanosoma brucei. In both cases the mitochondrial activity has been reduced to a minimum. The citric-acid cycle is not active and a classical electron-transport chain is absent. Both trypanosomes are unable to respire on praline, 2-oxoglutarate or succinate. We have described in Phytomonas sp. The presence of a mitochondrial alcohol dehydrogenase, which can explain the observed accumulation of ethanol as an end-product of glycolisis (Uttaro and Opperdoes, 1997, Mol. Biochem. Parasitol. 85: 213-219). However, the preferred substrates are isopropanol and acetone, for the forward and reverse reaction, respectively. This isopropanol dehydrogenase (iPDH) activity is present only in isolates of Phytomonas sp., which makes it a useful marker for the genus. We show here evidences that iPDH could be related to the amino-acid metabolism, which might be the only energy source for Phytomonas into the insect host. The enzyme was purified, and sequences of tryptic peptides obtained. By inverse genetic and screening of a genomic sublibrary, a fragment of 3800 pb was isolated, subcloned and sequenced, in order to identify the iPDH gene and putative associated genes.