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Protoporphyrinogen IX oxidase, a monotopic membrane protein, which catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX in the heme/chlorophyll biosynthetic pathway, is distributed widely throughout nature. Here we present the structure of protoporphyrinogen IX oxidase from Myxococcus xanthus, an enzyme with similar catalytic properties to human protoporphyrinogen IX oxidase that also binds the common plant herbicide, acifluorfen. In the native structure, the planar porphyrinogen substrate is mimicked by a Tween 20 molecule, tracing three sides of the macrocycle. In contrast, acifluorfen does not mimic the planarity of the substrate but is accommodated by the shape of the binding pocket and held in place by electrostatic and aromatic interactions. A hydrophobic patch surrounded by positively charged residues suggests the position of the membrane anchor, differing from the one proposed for the tobacco mitochondrial protoporphyrinogen oxidase. Interestingly, there is a discrepancy between the dimerization state of the protein in solution and in the crystal. Conserved structural features are discussed in relation to a number of South African variegate porphyria-causing mutations in the human enzyme.Protoporphyrinogen oxidase (PPOX) 5 (EC 1.3.3.4), the penultimate enzyme in the heme biosynthetic pathway, is the last common enzyme in heme and chlorophyll biosynthesis. It catalyzes the six-electron oxidation of protoporphyrinogen IX to protoporphyrin IX (1, 2). In humans, defects in the PPOX gene result in the dominantly inherited disorder variegate porphyria (VP) (3, 4), characterized by cutaneous photosensitivity and the propensity to develop acute neurovisceral crisis (5). This disease is particularly prevalent in South Africa because of a founder gene effect (an R59W mutation) (6, 7). In plants, PPOX is a target of light-dependent peroxidizing herbicides, which act as competitive inhibitors (8) resulting in desiccation and photobleaching of green plant tissue. Of the herbicides known to inhibit PPOX, the diphenylethers, of which acifluorfen (AF) is the most well studied, are of great interest. Such inhibition has also been documented in mammals and bacteria (9, 10). Inhibition of the enzyme leads to the accumulation and export of protoporphyrinogen to the cytoplasm, where it undergoes non-enzymatic oxidation to porphyrin. In the presence of light, accumulation of protoporphyrin can lead to reactive oxygen species that may induce cellular damage, primarily via peroxidation of membrane lipids, and ultimately cell death. A similar photodestructive mechanism, because of accumulating porphyrin intermediates in the skin of patients with VP, is probably responsible for the photocutaneous manifestations in this condition (11).In eukaryotes, PPOX is functionally conserved despite a low sequence identity. It is an intrinsic protein of the inner mitochondrial membrane and requires molecular oxygen and a flavin cofactor (in most cases, flavin adenine dinucleotide (FAD)) for this conversion. However, it is p...

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Pseudomonas aeruginosa depends on its quorum sensing (QS) system for its virulence factors' production and biofilm formation. Biofilms of P. aeruginosa on the surface of indwelling catheters are often resistant to antibiotic therapy. Alternative approaches that employ QS inhibitors alone or in combination with antibiotics are being developed to tackle P. aeruginosa infections. Here, we have studied the mechanism of action of 3-Phenyllactic acid (PLA), a QS inhibitory compound produced by Lactobacillus species, against P. aeruginosa PAO1. Our study revealed that PLA inhibited the expression of virulence factors such as pyocyanin, protease, and rhamnolipids that are involved in the biofilm formation of P. aeruginosa PAO1. Swarming motility, another important criterion for biofilm formation of P. aeruginosa PAO1, was also inhibited by PLA. Gene expression, mass spectrometric, functional complementation assays, and in silico data indicated that the quorum quenching and biofilm inhibitory activities of PLA are attributed to its ability to interact with P. aeruginosa QS receptors. PLA antagonistically binds to QS receptors RhlR and PqsR with a higher affinity than its cognate ligands N-butyryl-L-homoserine lactone (C-HSL) and 2-heptyl-3,4-dihydroxyquinoline (PQS; Pseudomonas quinolone signal). Using an in vivo intraperitoneal catheter-associated medaka fish infection model, we proved that PLA inhibited the initial attachment of P. aeruginosa PAO1 on implanted catheter tubes. Our in vitro and in vivo results revealed the potential of PLA as anti-biofilm compound against P. aeruginosa.

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