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

Abstract

ABSTRACT Pyruvate:quinone oxidoreductase catalyzes the oxidative decarboxylation of pyruvate to acetate and CO 2 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 Mg 2+ ( K m = 29 μM), Mn 2+ ( K m = 2 μM), and Co 2+ ( 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 .