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

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. In addition to several dyes (2,6-dichloroindophenol, p-iodonitrotetrazolium violet, and nitroblue tetrazolium), menadione (K m ‫؍‬ 106 M) 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.Corynebacterium glutamicum is an aerobic, gram-positive organism that grows on a variety of sugars and organic acids and is widely used in the industrial production of amino acids, particularly L-glutamate and L-lysine (40). Due to its importance for the carbon flux distribution within the metabolism and for the precursor supply for amino acid synthesis, the phosphoenolpyruvate (PEP)-pyruvate node of this organism (see Fig. 1) has been intensively studied and much attention has been focused on some of the enzymes involved, e.g., pyruvate kinase, PEP carboxylase, pyruvate carboxylase, and PEP carboxykinase (24,33,34,51,52,55). The oxidative decarboxylation of pyruvate and thus the fueling of the tricarboxylic acid (TCA) cycle with acetyl coenzyme A (acetyl-CoA) in C. glutamicum have been generally attributed to the pyruvate dehydrogenase complex (16,40,61).The genome of C. glutamicum has recently been determined and annotated (GenBank accession numbers NC_003450 and BX927147) (35,63), and an open reading frame (cg2891) with significant identity to the Escherichia coli pyruvate oxidase gene (poxB) has been detected (8, 35). This finding indicated the presence of an additional pyruvate-decarboxylating enzyme at the C. glutamicum PEP-pyruvate node (Fig. 1). The E. coli pyruvate oxidase (EC 1.2.2.2) catalyzes the oxidative decarboxylation of pyruvate to acetate and CO 2 (68) and is a nonessential, peripheral membrane protein consisting of four identical subunits each containing tightly bound flavin adenine dinucleotide (FAD) and loosely bound thiamine pyrophosphate (TPP) and Mg 2ϩ (6,26,43,44,68). As the reaction of the enzyme is oxygen independent and uses ubi...

Pyruvate:Quinone Oxidoreductase in Corynebacterium glutamicum : Molecular Analysis of the pqo Gene, Significance of the Enzyme, and Phylogenetic Aspects

“…Like the E. coli enzyme, the C. glutamicum PQO is a homotetrameric flavoprotein containing TPP, and it is activated by Triton X-100, phosphatidylglycerol and dipalmitoyl-phosphatidylglycerol. In contrast, it does not reduce ferricyanide, which is routinely used for assaying the E. coli enzyme (1,9,75), and it probably uses a menaquinone instead of ubiquinone as the physiological electron acceptor (59). The C. glutamicum PQO activity was highest in cells grown on complex medium and about threefold lower when glucose was added to the complex medium or when the cells were grown on minimal medium containing different carbon sources, suggesting that the enzyme is regulated by the carbon source in the growth medium.…”

“…Very recently, we showed for the first time the presence of PQO activity in a prokaryotic organism apart from E. coli and described the isolation, purification, and biochemical analysis of the PQO enzyme from Corynebacterium glutamicum (59). This bacterium is an aerobic, "high-GC gram-positive" organism that grows on a variety of sugars and organic acids and is widely used in the industrial production of amino acids, particularly L-glutamate and L-lysine (35).…”

“…This bacterium is an aerobic, "high-GC gram-positive" organism that grows on a variety of sugars and organic acids and is widely used in the industrial production of amino acids, particularly L-glutamate and L-lysine (35). Due to its importance for the distribution of the carbon flux within the metabolism and for the precursor supply for amino acid synthesis, the phosphoenolpyruvate-pyruvate node of this organism has been intensively studied (reviewed in reference 19), and much attention has been focused on pyruvate-converting enzymes, such as pyruvate carboxylase (34,49,50,51), pyruvate dehydrogenase complex (14,60,66), and PQO (59). Like the E. coli enzyme, the C. glutamicum PQO is a homotetrameric flavoprotein containing TPP, and it is activated by Triton X-100, phosphatidylglycerol and dipalmitoyl-phosphatidylglycerol.…”

Flavin Redox Switching of Protein Functions

“…According to a recent phylogenetic survey of PQO type (e.g., EcPOX) and POX type (e.g., LpPOX) enzymes, it appears that they are interspersed in gram positive and gram negative bacteria lineages (62). In the same study, physiological experiments with Corynebacterium glutamicum, a gram-positive aerobe expressing a PQO type enzyme, suggested that this enzyme is regulated by carbon sources rather than growth phase as observed for EcPOX (61,62). The authors concluded that the difference in the surrounding genetic makeup of the C. glutamicum pqo gene compared to the poxB gene in E. coli were most likely responsible for the regulational differences (61,62).…”

“…In the same study, physiological experiments with Corynebacterium glutamicum, a gram-positive aerobe expressing a PQO type enzyme, suggested that this enzyme is regulated by carbon sources rather than growth phase as observed for EcPOX (61,62). The authors concluded that the difference in the surrounding genetic makeup of the C. glutamicum pqo gene compared to the poxB gene in E. coli were most likely responsible for the regulational differences (61,62). With a paucity of literature on PQO type enzymes from organisms other than E. coli, the remainder of this section will focus on EcPOX.…”