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Journal of Bacteriology, February 2005, p. 862-871, Vol. 187, No. 3
0021-9193/05/$08.00+0     doi:10.1128/JB.187.3.862-871.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Pyruvate:Quinone Oxidoreductase from Corynebacterium glutamicum: Purification and Biochemical Characterization

Department of Microbiology and Biotechnology, University of Ulm, Ulm, Germany¹

Received 8 July 2004/ Accepted 28 October 2004

Pyruvate:quinone oxidoreductase catalyzes the oxidative decarboxylation of pyruvate to acetate and CO₂ with a quinone as the physiological electron acceptor. So far, this enzyme activity has been found only in Escherichia coli. Using 2,6-dichloroindophenol as an artificial electron acceptor, we detected pyruvate:quinone oxidoreductase activity in cell extracts of the amino acid producer Corynebacterium glutamicum. The activity was highest (0.055 ± 0.005 U/mg of protein) in cells grown on complex medium and about threefold lower when the cells were grown on medium containing glucose, pyruvate, or acetate as the carbon source. From wild-type C. glutamicum, the pyruvate:quinone oxidoreductase was purified about 180-fold to homogeneity in four steps and subjected to biochemical analysis. The enzyme is a flavoprotein, has a molecular mass of about 232 kDa, and consists of four identical subunits of about 62 kDa. It was activated by Triton X-100, phosphatidylglycerol, and dipalmitoyl-phosphatidylglycerol, and the substrates were pyruvate (k_cat = 37.8 ± 3 s^–1; K_m = 30 ± 3 mM) and 2-oxobutyrate (k_cat = 33.2 ± 3 s^–1; K_m = 90 ± 8 mM). Thiamine pyrophosphate (K_m = 1 µM) and certain divalent metal ions such as Mg²⁺ (K_m = 29 µM), Mn²⁺ (K_m = 2 µM), and Co²⁺ (K_m = 11 µM) served as cofactors. In addition to several dyes (2,6-dichloroindophenol, p-iodonitrotetrazolium violet, and nitroblue tetrazolium), menadione (K_m = 106 µM) was efficiently reduced by the purified pyruvate:quinone oxidoreductase, indicating that a naphthoquinone may be the physiological electron acceptor of this enzyme in C. glutamicum.

This article has been cited by other articles:

• Schreiner, M. E., Riedel, C., Holatko, J., Patek, M., Eikmanns, B. J. (2006). Pyruvate:Quinone Oxidoreductase in Corynebacterium glutamicum: Molecular Analysis of the pqo Gene, Significance of the Enzyme, and Phylogenetic Aspects. J. Bacteriol. 188: 1341-1350 [Abstract] [Full Text]  
• Schreiner, M. E., Fiur, D., Holatko, J., Patek, M., Eikmanns, B. J. (2005). E1 Enzyme of the Pyruvate Dehydrogenase Complex in Corynebacterium glutamicum: Molecular Analysis of the Gene and Phylogenetic Aspects. J. Bacteriol. 187: 6005-6018 [Abstract] [Full Text]