Sulfhydryl Chemistry Detects Three Conformations of the Lipid Binding Region of Escherichia coli Pyruvate Oxidase

Abstract

Site-specific disulfide cross-linking experiments detected a conformational change within the C-terminal segment of Escherichia coli pyruvate oxidase (PoxB), a lipid-activated homotetrameric enzyme, upon substrate binding [Chang, Y.-Y., & Cronan, J. E., Jr. (1995) J. Biol. Chem. 270, 7896-7901]. The C-terminal lipid binding regions were cross-linked only in the presence of the substrate, pyruvate, and the thiamine pyrophosphate cofactor, indicating close proximity of a pair of C termini. We have now systematically substituted cysteine at 18 additional amino acid positions within the C-terminal region to obtain a panel of 21 proteins each having a single residue changed to cysteine. These proteins have been studied by disulfide cross-linking and by accessibility of the cysteine side chain to a variety of sulfhydryl agents. In the absence of pyruvate, the cysteine residues of the modified PoxB proteins failed to form disulfide bonds, generally failed to react with a large and rigid hydrophilic sulfhydryl reagent, 4-acetamido-4'-[(iodoacetyl)amino]stilbene-2,2'-disulfonic acid (IASD), and in some cases reacted weakly with a smaller more hydrophobic reagent, N-ethylmaleimide. Therefore, in this conformation, the C termini appear fixed in a rigid environment having limited exposure to solvent. In the presence of pyruvate, all of the C-terminal cysteine residues (except the two most distal from the C terminus) reacted with both sulfhydryl reagents and readily formed disulfide cross-linked species, indicating conversion to a structure having a high degree of conformational freedom. In the presence of lipid activators, Triton X-100 or dipalmitoylphosphatidylglycerol, a subset of the cysteine-substituted proteins no longer reacted with the membrane-impermeable IASD reagent, indicating penetration of these protein segments into the lipid micelles. For most of the proteins, similar extents of disulfide formation were seen upon addition of an oxidizing agent in the presence or absence of lipid activators. An exception was PoxB D560C which was much more readily cross-linked in the presence of lipid. Moreover, a subset of PoxB proteins that cross-linked to lower extents in the presence of lipids was found. The behavior of these proteins provides strong support for the model in which two C termini associate to form the functional lipid binding domain. These data are discussed in terms of three distinct PoxB conformers and the known crystal structure of a highly related protein.

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

“…The binding to Triton X-114 was completely reversible and dependent on the presence of the substrate pyruvate and the cofactors TPP and Mg 2ϩ . These data suggest that, analogous to the situation shown for the E. coli pyruvate oxidase (14,44,53,58), the C. glutamicum pyruvate:quinone oxidoreductase may undergo a conformational change upon incubation with a substrate and cofactors, resulting in the exposure of an enzyme domain that enables in vitro the binding of pyruvate:quinone oxidoreductase to the hydrophobic detergent Triton X-114 or in vivo the binding of the enzyme to the membrane. The E. coli pyruvate oxidase is thought to shuttle between the cytosol and the inner membrane, depending on the intracellular pyruvate concentration (11,15).…”

“…The enzyme has been shown to be strongly activated by low concentrations of phospholipids and detergents (5,11,20,21,29), resulting in an increase of about 20-fold in V max and a decrease of about 10-fold in the K m for pyruvate (26,43). The (phospho)lipid activation by and association with the protein are accompanied by conformational changes and alteration of various properties of the enzyme (13,14,67). Expression of the E. coli pyruvate oxidase gene (poxB) was shown to be induced in the stationary growth phase and to be dependent on sigma factor RpoS (10).…”

Pyruvate oxidase contributes to the aerobic growth efficiency of Escherichia coli

“…Moreover, this activation can be mimicked by limited chymotryptic proteolysis leading to the cleavage of a 3 kDa peptide from the C-terminus of the enzyme (Russell et al, 1977 ;Recny et al, 1985). The poxB gene has been cloned and sequenced (Grabau & Cronan, 1984, 1986a and this has greatly facilitated recent studies on both the structure-function relationships (including the lipid activation) of PoxB (Grabau et al, 1989 ;Wang et al, 1991 ;Chang & Cronan, 1997) and on the transcriptional regulation of the poxB gene (Chang et al, 1994). The regulatory studies indicated that PoxB activity and poxB-lacZ expression reach maximal values in early stationary phase and both are completely dependent on the rpoS-encoded sigma factor (RpoS, σ$) or σ S ) that is required for transcribing many genes that are induced in stationary phase.…”

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

“…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).…”