Crystal Structure of Protoporphyrinogen Oxidase from Myxococcus xanthus and Its Complex with the Inhibitor Acifluorfen

Abstract

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...

Mutations in hemG Mediate Resistance to Salicylidene Acylhydrazides, Demonstrating a Novel Link between Protoporphyrinogen Oxidase (HemG) and Chlamydia trachomatis Infectivity

“…7B). BsPPO has recently been cocrystalized with flavine-adenine dinucleotide (FAD) and the inhibitor acifluorfen (AF); the latter is proposed to mimic half of the PPO substrate protoporphyrinogen XI and therefore binds to the active site of BsPPO (48,49). This structural information allowed us to map the mutations that confer IS-INP0341 resistance onto a structural model of the Chlamydia HemG.…”

Noncanonical coproporphyrin-dependent bacterial heme biosynthesis pathway that does not use protoporphyrin

“…A crystal structure exists for this protein (29), which is of a similar size and structure as the eukaryotic PpoX (30,31) and Gram-negative HemY (32). However, it possesses four distinctions from these enzymes: (i) it is a soluble monomer, (ii) it is relatively insensitive to the herbicide acifluorfen, (iii) it is unable to complement a ΔppoX (hemG) mutant of E. coli, and (iv) it is able to oxidize coproporphyrinogen to coproporphyrin.…”

Discovery of a Gene Involved in a Third Bacterial Protoporphyrinogen Oxidase Activity through Comparative Genomic Analysis and Functional Complementation

“…Some bacteria, such as Myxococcus and Aquifex, possess an oxygen-dependent, membrane-associated enzyme, HemY, which is homologous to eukaryotic protoporphyrinogen oxidase (PPOX; the general abbreviation for protoporphyrinogen oxidase is PPO) (6,7,50). Firmicutes and Actinobacteria contain a soluble version of HemY that is different in that it lacks the membrane binding domain (8) and the enteric Gammaproteobacteria employ the oxygen-independent enzyme HemG (20,44), which is a quinone-dependent flavodoxin with protoporphyrinogen dehydrogenase (PpdH) activity that can be coupled to aerobic or anaerobic respiration (2,34).…”