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

Abstract

Corynebacterium glutamicum recently has been shown to possess pyruvate:quinone oxidoreductase (PQO), catalyzing the oxidative decarboxylation of pyruvate to acetate and CO 2 with a quinone as the electron acceptor. Here, we analyze the expression of the C. glutamicum pqo gene, investigate the relevance of the PQO enzyme for growth and amino acid production, and perform phylogenetic studies. Expression analyses revealed that transcription of pqo is initiated 45 bp upstream of the translational start site and that it is organized in an operon together with genes encoding a putative metal-activated pyridoxal enzyme and a putative activator protein. Inactivation of the chromosomal pqo gene led to the absence of PQO activity; however, growth and amino acid production were not affected under either condition tested. Introduction of plasmid-bound pqo into a pyruvate dehydrogenase complex-negative C. glutamicum strain partially relieved the growth phenotype of this mutant, indicating that high PQO activity can compensate for the function of the pyruvate dehydrogenase complex. To investigate the distribution of PQO enzymes in prokaryotes and to clarify the relationship between PQO, pyruvate oxidase (POX), and acetohydroxy acid synthase enzymes, we compiled and analyzed the phylogeny of respective proteins deposited in public databases. The analyses revealed a wide distribution of PQOs among prokaryotes, corroborated the hypothesis of a common ancestry of the three enzymes, and led us to propose that the POX enzymes of Lactobacillales were derived from a PQO.Pyruvate:quinone oxidoreductase (PQO) (EC 1.2.2.2) catalyzes the oxidative decarboxylation of pyruvate to acetate and CO 2 with a quinone as the physiological electron acceptor. The Escherichia coli PQO enzyme, also designated pyruvate oxidase (POX), 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 (4,22,37,38,75). This enzyme has been shown to be strongly activated by low concentrations of phospholipids and detergents (5,8,15,16,28), and the activation has been shown to be accompanied by conformational changes and alteration of various properties of the enzyme (11,12,74). Aside from extensive biochemical characterization of the E. coli PQO, the respective gene ( poxB) and its expression have been studied in detail (7, 9, 25). Expression of the poxB gene was dependent on sigma factor RpoS and thus induced in the stationary growth phase (7). Accordingly, the authors speculated that the enzyme might be responsible for oxidative pyruvate decarboxylation during the transition between the exponential aerobic growth phase and the stationary growth phase when cultures become anaerobic. Further studies with poxB inactivation mutants and with poxBoverexpressing strains of E. coli indicated that PQO activity contributes to the aerobic growth efficiency and that a high PQO activity, together with acetyl-coenzyme A (CoA) syn...

Flavin Redox Switching of Protein Functions

“…Even though EcPOX is called a pyruvate ''oxidase'', it is well established that the physiological electron acceptor is membrane-bound ubiquinone-8 (10,30,42). Therefore, this enzyme has been more accurately referred to as a pyruvate:quinone oxidoreductase (PQO) (62). In contrast, other prokaryotic POX enzymes readily use O 2 as the electron acceptor and are thus true oxidases generating H 2 O 2 during every catalytic cycle (62).…”

“…Therefore, this enzyme has been more accurately referred to as a pyruvate:quinone oxidoreductase (PQO) (62). In contrast, other prokaryotic POX enzymes readily use O 2 as the electron acceptor and are thus true oxidases generating H 2 O 2 during every catalytic cycle (62). One well-studied example is POX from Lactobacillus plantarum (LpPOX) (49,79).…”

“…LpPOX not only differs from EcPOX in oxygen reactivity, but also uses P i to produce acetylphosphate (49). Very little is known about how these aforementioned differences relate to the physiological and regulatory functions of POX enzymes (62). 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).…”

“…Members of the POX enzyme superfamily are distinguished by well-defined catalytic and structural differences even among prokaryotic enzymes (62). For example, POX enzymes can be divided according to the natural electron acceptor employed in the POX reaction cycle (62).…”

“…For example, POX enzymes can be divided according to the natural electron acceptor employed in the POX reaction cycle (62). Even though EcPOX is called a pyruvate ''oxidase'', it is well established that the physiological electron acceptor is membrane-bound ubiquinone-8 (10,30,42).…”