Molecular cloning, DNA sequencing, and enzymatic analyses of two Escherichia coli pyruvate oxidase mutants defective in activation by lipids

Abstract

Two Escherichia coli pyruvate oxidase (EC 1.2.2.2) mutant genes, poxB3 and poxB4, were cloned on plasmid pBR322. The poxB3 mutant oxidase which was described previously (Y. Y. Chang and J. E. Cronan, Jr., Proc. Natl. Acad. Sci. USA 81: [4348][4349][4350][4351][4352] 1984) was deficient in lipid activation but retained full catalytic activity.The poxB3 mutation was located in the C-terminal half of the gene, and the nucleotide alteration has been determined by DNA sequencing of this part of the gene and by comparing the sequence with that of the wild-type strain (C. Grabau and J. E. Cronan, Jr., submitted for publication). The poxB3 oxidase mutation is the substitution of a serine residue for Pro-536. poxB4, another pyruvate oxidase mutant gene, was also deficient in lipid activation. The major difference between the poxB3 and poxB4 oxidase was in the binding of Triton detergents. The poxB4 mutation was also located in the C-terniinal half of the gene, and sequence analysis has shown that only one nucleotide base was altered, which resulted in Ala-467 being converted to a threonine residue. The results of the amino acid substitutions in the mutant proteins, leading to the functional alteration of the enzyme, are discussed. Preparation of plasmids, DNA sequencing, and other methods involving DNA. Purified plasmids were isolated and prepared by the mild Triton X-100-lysozyme lysis of chloramphenicol-amplified cultures (22) followed by two cycles of cesium chloride-ethidium bromide gradient centrifugation (21). For strains which are lysed poorly by this method (e.g., CG3), the large-scale alkaline lysis method (21) was used.

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

“…Furthermore, residue Gly35 in the Lactobacillus plantarum sequence, suggested to be a possible binding site for inorganic phosphate (47), is also unique to the lactobacterial pyruvate oxidases and is replaced by aspartate residues in the phosphate-independent pyruvate:quinone oxidoreductase enzymes. In contrast, a proline residue (Pro536) shown to be essential for lipid activation of the E. coli pyruvate oxidase (12) is conserved in all but the lactobacterial enzymes shown in Fig. 2.…”

Structural basis for membrane binding and catalytic activation of the peripheral membrane enzyme pyruvate oxidase from Escherichia coli

“…The structure thus reveals that the C-terminal domain is held firmly in place by the half-barrel super secondary structure and a large number of hydrogen-bonding and electrostatic interactions evenly distributed over the whole sequence. Earlier mutagenesis data had already indicated that several residues of the C terminus (E 564 , R 572 ) and residues of the active center loop (A 467 ) interacting with the half-barrel motif play critical roles for membrane binding and activity (29,30). Most remarkably, many of the residues implicated in forming the proposed membrane-binding amphipathic helix (G 559 -D-E-V-I-E-L-A-K-T 568 ) are located in the sheet structure.…”

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.…”

“…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).…”