Acylation stabilizes a protease-resistant conformation of protoporphyrinogen oxidase, the molecular target of diphenyl ether-type herbicides

Abstract

Protoporphyrinogen oxidase is a mitochondrial membranebound flavoprotein that catalyzes the penultimate reaction in the heme biosynthesis pathway, the oxygen-dependent aromatization of protoporphyrinogen IX to protoporphyrin IX (1). This enzyme has been shown to be the molecular target of diphenyl ether-type herbicides in plants (2, 3). The biochemical characteristics of the protein are those of an intrinsic protein, but sequence analysis of cloned yeast, mammalian, or plant protoporphyrinogen oxidases failed to reveal any typical membrane-spanning ␣-helical structure (4-9). Studies of the biogenesis of the yeast enzyme have shown that the protein is synthesized as a precursor that is rapidly converted to the active form of protoporphyrinogen oxidase, but that this maturation does not involve the removal of a N-terminal mitochondrial targeting peptide (6, 10). Although the sequence of protoporphyrinogen oxidase contains several hydrophobic regions, none of those is longer than 15 uncharged residues, and they are therefore unlikely to form membrane spanning segments; the largest hydrophobic domain in the protein is very homologous to the ␤␣␤-dinucleotide (Rossman)-binding fold found in many flavoproteins. Anchoring to the membrane may involve amphipathic helical domains that could be responsible for insertion of the protoporphyrinogen oxidase into the inner mitochondrial membrane, as described for prostaglandin H 2 synthase-1, a monotopic membrane-bound protein of the endoplasmic reticulum (11). The conserved domain A in protoporphyrinogen oxidases (12) (Fig. 1A) may well represent such an anchoring structure, and hydrophobic cluster analysis (13) of this domain (Fig. 1B) shows striking similarities to the membrane-anchoring domain of prostaglandin H 2 synthase (Fig. 1C). Another possibility, compatible with a posttranslational modification of the protein leading to the shift in electrophoretic mobility initially attributed to the proteolytic cleavage of a putative presequence previously described (10), is that protoporphyrinogen oxidase is anchored to the inner mitochondrial membrane by a different mechanism, such as acylation. This prompted us to look for a potential posttranslational modification of the polypeptide chain that could contribute to both the highly hydrophobic nature of the protein and the difference in electrophoretic mobility of the precursor and the mature forms of the protein. Studies on the biogenesis of the Ras oncogene products showed that acylation of these proteins may alter their electrophoretic mobility and hydrophobicity (14). We therefore investigated the possibility that protoporphyrinogen oxidase is an acylated protein. Materials and MethodsMaterials. Protoporphyrin IX, disodium salt, was purchased from Serva. l-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (EC 3.4.21.4) from bovine pancreas used either as a soluble enzyme (Type XIII, 10,000-13,000 units⅐mg Ϫ1 ) or attached to agarose beads (75-100 units⅐ml Ϫ1 packed gel), cerulenin (2, 3-epoxy-4-oxo-7, 10 dodecadie...

Examination of mitochondrial protein targeting of haem synthetic enzymes: in vivo identification of three functional haem-responsive motifs in 5-aminolaevulinate synthase

“…Whereas some computer algorithms predict the N-terminal dinucleotide-binding motif as a transmembrane segment, it is clear from analogy with a number of other FAD-containing proteins that this motif does not participate in membrane attachment [26]. Indeed, there is no evidence that PPO has a transmembrane motif and it has been proposed that a proteinbound lipid mediates membrane attachment [11], although the recently published structure does not show the presence of a lipid in the enzyme from tobacco [12].…”

“…Association with the membrane has been proposed to involve a protein-bound fatty acid [11], although recently it has been suggested that PPO forms a transmembrane complex with FC [12]. This homodimeric protein does not contain a typical N-terminal targeting sequence and is not proteolytically processed, but does have a dinucleotide-binding motif at the N-terminal end that is essential for binding of the enzyme's cofactor, FAD [13].…”

N-Terminal Fatty Acid Substitution Increases the Leishmanicidal Activity of CA(1-7)M(2-9), a Cecropin-Melittin Hybrid Peptide

“…The acylation process is enzymatic, with a variable degree of specificity for both the fatty acid and the primary structure of the protein (17). A major role for protein acylation is to increase membrane association, although other effects such as improved proteolytic stability (6) and sorting into specific subcellular localizations have also been described (30,38).…”

“…Therapy based on the rational use of such peptides appears to be a feasible alternative, and successful preliminary results have been reported for natural peptides such as dermaseptins (19,23), SPYY (47), cecropin A (1), and gomesin (42), all of which have been shown to be active against different forms of the parasite. In our own work, we have expanded the repertoire of peptides with activity against Leishmania with cecropin-melittin hybrids such as CA (1)(2)(3)(4)(5)(6)(7)(8)M (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) and CA(1-7)M(2-9), both of which are active in the micromolar range (15).…”

Straightforward Method for the Preparation of Lysine-Based Double-Chained Anionic Surfactants

“…[1][2][3] Thus, the increase of protein's lipophilicity conferred by fatty-acid acylation can, among other effects, (i) increase the protein's stability against proteolytic degradation, (ii) target the protein for certain intracellular compartments, and (iii) promote the protein's interaction and association with phospholipid bilayers in cell membranes. [1][2][3][4][5][6] This latter feature of lipoproteins has been the driving force for N-fatty-acid acylation of antimicrobial peptide's N-terminal and=or lysine residues as a strategy to improve their antibiotic activity. [7][8][9][10][11][12][13][14][15][16] However, the potential interest of N-acylation of proteinogenic amino acids with natural fatty acids to produce surface-active agents of gemini type has been little explored, despite the urgent need for new double-chain surfactants that are simultaneously efficient detergents with anti bacterial properties, biodegradable, and biocompatible.…”