TAILIEUCHUNG - Báo cáo Y học: Purification, characterization and subunits identification of the diol dehydratase of Lactobacillus collinoides

The three genespduCDEencoding the diol dehydratase of Lactobacillus collinoides, have been cloned for overexpres-sion in the pQE30 vector. Although the three subunits of the protein were highly induced, no activity was detected in cell extracts. The enzyme was therefore purified to near homo-geneity by ammoniumsulfate precipitation and gel filtration chromatography. In fractions showing diol dehydratase activity, three main bands were present after SDS/PAGE with molecular masses of 63, 28 and 22 kDa, respectively | Eur. J. Biochem. 269 5731-5737 2002 FEBS 2002 doi Purification characterization and subunits identification of the diol dehydratase of Lactobacillus collinoides Nicolas Sauvageot1 Vianney Pichereau1 Lo ic Louarme2 Axel Hartke1 Yanick Auffray1 and Jean-Marie Laplace1 1USC INRA de Microbiologie de l Environnement Université de Caen France 2Chaire de Biochimie Industrielle et Agro-Alimentaire CNAM Paris France The three genes pduCDE encoding the diol dehydratase of Lactobacillus collinoides have been cloned for overexpression in the pQE30 vector. Although the three subunits of the protein were highly induced no activity was detected in cell extracts. The enzyme was therefore purified to near homogeneity by ammonium sulfate precipitation and gel filtration chromatography. In fractions showing diol dehydratase activity three main bands were present after SDS PAGE with molecular masses of 63 28 and 22 kDa respectively. They were identified by mass spectrometry to correspond to the large medium and small subunits of the dehydratase encoded by the pduC pduD and pduE genes respectively. The molecular mass of the native complex was estimated to 207 kDa in accordance with the calculated molecular masses deduced from the pduC D E genes 61 and 19 1 kDa respectively and a a2b2y2 composition. The Km for the three main substrates were mM for 1 2-propanediol mM for 1 2-ethanediol and mM for glycerol. The enzyme required the adenosylcobalamin coenzyme for catalytic activity and the Km for the cofactor was 8 M. Inactivation of the enzyme was observed by both glycerol and cyanocobalamin. The optimal reaction conditions of the enzyme were pH and 37 C. Activity was inhibited by sodium and calcium ions and to a lesser extent by magnesium. A fourth band at 59 kDa copurified with the diol dehydratase and was identified as the propionaldehyde dehydrogenase enzyme another protein involved in the 1 2-propanediol metabolism pathway. .

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