Pyruvate:quinone oxidoreductase from Corynebacterium glutamicum: purification and biochemical characterization

Abstract

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 (kcat=37.8 +/- 3 s(-1); Km=30 +/- 3 mM) and 2-oxobutyrate (kcat=33.2 +/- 3 s(-1); Km=90 +/- 8 mM). Thiamine pyrophosphate (Km=1 microM) and certain divalent metal ions such as Mg2+ (Km=29 microM), Mn2+ (Km=2 microM), and Co2+ (Km=11 microM) served as cofactors. In addition to several dyes (2,6-dichloroindophenol, p-iodonitrotetrazolium violet, and nitroblue tetrazolium), menadione (Km=106 microM) 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.

FIG. 1.

PEP-pyruvate node in C. glutamicum. Abbreviations: AK, acetate kinase; PCx, pyruvate carboxylase; PDHC, pyruvate dehydrogenase complex; PEPCk, PEP carboxykinase; PEPCx, PEP carboxylase; PK, pyruvate kinase; PQO, pyruvate:quinone oxidoreductase; PTA, phosphotransacetylase.

FIG. 2.

Sequence alignment of the pyruvate:quinone oxidoreductases or pyruvate oxidases of C. glutamicum (accession number NP_601811), Streptomyces coelicolor (T34668), E. coli (NP_415392), Salmonella enterica serovar Typhimurium (S_typhimurium; NP_459912), Bradyrhizobium japonicum (NP_773926), Lactobacillus plantarum (NP_786788), Lactococcus lactis (NP_26820), and Streptococcus pneumoniae (AAB40976). Amino acids identical in at least seven sequences are shaded in grey, and amino acids identical in at least five sequences are shaded in black. The TPP signature motif is underlined.

FIG. 3.

Growth (squares) and pyruvate:quinone oxidoreductase (PQO) activities (bars) of WT C. glutamicum grown on TY medium without glucose (closed symbols) or with 4% glucose (open symbols). The standard deviations were in all cases below 10%.

FIG. 4.

SDS-PAGE of pyruvate:quinone oxidoreductase from C. glutamicum after each step of the purification procedure. Lane 1, crude extract; lane 2, extract after ultracentrifugation; lane 3, extract after heat denaturation; lane 4, purified pyruvate:quinone oxidoreductase. Proteins were visualized by silver staining.