Expression and characterization of the terminal heme synthetic enzymes from the hyperthermophile<i>Aquifex aeolicus</i>

Wang, Kai-Fen; Dailey, Tamara A.; Dailey, Harry A.

doi:10.1111/j.1574-6968.2001.tb10789.x

Cited by 15 publications

(4 citation statements)

References 16 publications

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“…The enzyme of the hyperthermophile A. aeolicus (263), however, has been reported to be membrane associated but monomeric. Unlike CgoX of Gram-positive bacteria, these enzymes use protoporphyrinogen IX, but not coproporphyrinogen III, as the substrate and are inhibited by the diphenyl ether herbicide acifluorfen at a 1 M concentration (261,263). The reaction mechanism of this enzyme appears identical to that of eukaryotic Ppox and Gram-positive CgoX enzymes (Fig.…”

Section: Prokaryotic Heme Biosynthesismentioning

confidence: 90%

“…Diverse but limited biochemical studies have been performed on the Gram-negative protoporphyrin ferrochelatases from R. sphaeroides (289, 290), A. itersonii (291), B. japonicum (292), Caulobacter crescentus (293), M. xanthus, Pseudomonas putida, Bdellovibrio bacteriovorus (228), and A. aeolicus (263). In general, these enzymes all utilize Co 2ϩ ,Z n 2ϩ ,N i 2ϩ , and Fe 2ϩ as metal substrates and deutero-, meso-, hemato-, and protoporphyrin IX as the porphyrin substrates.…”

Section: Metalation Of Protoporphyrin IX By Protoporphyrin Ferrochelamentioning

confidence: 99%

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Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product

Dailey

Gerdes³

et al. 2017

Microbiol Mol Biol Rev

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SUMMARY The advent of heme during evolution allowed organisms possessing this compound to safely and efficiently carry out a variety of chemical reactions that otherwise were difficult or impossible. While it was long assumed that a single heme biosynthetic pathway existed in nature, over the past decade, it has become clear that there are three distinct pathways among prokaryotes, although all three pathways utilize a common initial core of three enzymes to produce the intermediate uroporphyrinogen III. The most ancient pathway and the only one found in the Archaea converts siroheme to protoheme via an oxygen-independent four-enzyme-step process. Bacteria utilize the initial core pathway but then add one additional common step to produce coproporphyrinogen III. Following this step, Gram-positive organisms oxidize coproporphyrinogen III to coproporphyrin III, insert iron to make coproheme, and finally decarboxylate coproheme to protoheme, whereas Gram-negative bacteria first decarboxylate coproporphyrinogen III to protoporphyrinogen IX and then oxidize this to protoporphyrin IX prior to metal insertion to make protoheme. In order to adapt to oxygen-deficient conditions, two steps in the bacterial pathways have multiple forms to accommodate oxidative reactions in an anaerobic environment. The regulation of these pathways reflects the diversity of bacterial metabolism. This diversity, along with the late recognition that three pathways exist, has significantly slowed advances in this field such that no single organism's heme synthesis pathway regulation is currently completely characterized.

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Section: Prokaryotic Heme Biosynthesismentioning

confidence: 90%

Section: Metalation Of Protoporphyrin IX By Protoporphyrin Ferrochelamentioning

confidence: 99%

Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product

Dailey

Gerdes³

et al. 2017

Microbiol Mol Biol Rev

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265

335

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“…Prokaryotic diversity with respect to the electron acceptor/donor used also appears to exist (see above). PPOX from the thermophile Aquifex aeolicus is most similar to M. xanthus differing only in its monomeric state (20).…”

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confidence: 99%

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

Corradi

Corrigall

Boix

et al. 2006

Journal of Biological Chemistry

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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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“…As the B. subtilis ferrochelatase is soluble, it differs from most other ferrochelatases, which are membrane-associated. [11,13] Our mutants, with increased in vivo activity based on certain structural or electrostatic changes at the surface of the B. subtilis ferrochelatase (Figure 3, Table 1), might therefore reflect a membrane or protein±protein interaction, which possibly enhances activity. + at m/z = 616.3) and proto IX (peak 2, l max = 505, 539, 574, and 628 nm; [M+H] + at m/z = 563.4).…”

Section: Resultsmentioning

confidence: 96%

A High‐Throughput Screen for Porphyrin Metal Chelatases: Application to the Directed Evolution of Ferrochelatases for Metalloporphyrin Biosynthesis

et al. 2004

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Porphyrins are of particular interest in a variety of applications ranging from biocatalysis and chemical synthesis to biosensor and electronic technologies as well as cancer treatment. Recently, we have developed a versatile system for the high-level production of porphyrins in engineered E. coli cells with the aim of diversifying substitution patterns and accessing porphyrin systems not readily available through chemical synthesis. However, this approach failed to produce significant amounts of the metalloporphyrin in vivo from overproduced protoporphyrin due to insufficient metal insertion. Therefore, we systematically assessed the activity of the B. subtilis ferrochelatase in vivo and in vitro. A true high-throughput-screening approach based on catalytic in vivo ferrochelatase activity was developed by using fluorescence-activated cell sorting (FACS). This assay was used to screen a library of 2.4 x 10(6) ferrochelatase mutants expressed in protoporphyrin-overproducing recombinant E. coli cells. Several selected protein variants were purified, and their improved catalytic activity was confirmed in vitro. In addition to ferrochelatase activity, metal transport into E. coli was identified as another limitation for in vivo heme overproduction. Overexpression of the metal transporter zupT as part of the assembled pathway increased the overall metalloporphyrin production twofold. This report represents the most exhaustive in vitro evolution study of a ferrochelatase and demonstrates the effectiveness of our novel high-throughput-screening system for directed evolution of ferrochelatases based on their catalytic activity.

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Expression and characterization of the terminal heme synthetic enzymes from the hyperthermophileAquifex aeolicus

Cited by 15 publications

References 16 publications

Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product

Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product

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

A High‐Throughput Screen for Porphyrin Metal Chelatases: Application to the Directed Evolution of Ferrochelatases for Metalloporphyrin Biosynthesis

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