Purification, characterization and subunits identification of the diol dehydratase of Lactobacillus collinoides.
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, 24.7 and 19,1 kDa, respectively) and a alpha2beta2gamma2 composition. The Km for the three main substrates were 1.6 mm for 1,2-propanediol, 5.5 mm for 1,2-ethanediol and 8.3 mm for glycerol. The enzyme required the adenosylcobalamin coenzyme for catalytic activity and the Km for the cofactor was 8 micro m. Inactivation of the enzyme was observed by both glycerol and cyanocobalamin. The optimal reaction conditions of the enzyme were pH 8.75 and 37 degrees 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.
Sauvageot
Nicolas
N
USC INRA de Microbiologie de l'Environnement, Université de Caen, France; Chaire de Biochimie Industrielle et Agro-Alimentaire, CNAM, Paris, France. phdlme@ibba.unicaen.fr
Pichereau
Vianney
V
Louarme
Loïc
L
Hartke
Axel
A
Auffray
Yanick
Y
Laplace
Jean-Marie
JM
D002850:Chromatography, Gel; D003001:Cloning, Molecular; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005990:Glycerol / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology*; D011408:Propanediol Dehydratase / Q000737:chemistry* / Q000302:isolation & purification*; D019946:Propylene Glycol / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors
D019946:Propylene Glycol; D011408:Propanediol Dehydratase; D005990:Glycerol