Subunit composition of pro-phenol oxidase from Manduca sexta: molecular cloning of subunit ProPO-P1

Phenol oxidase (PO) is known to play an important role in defense mechanisms in insect immunity. It is present as a zymogen in insect hemolymph, and can be activated by a specific proteolytic reaction that is stimulated by microbial cell wall components. The pro-phenol oxidase (pro-PO) purified from the larval hemolymph of Manduca sexta contains two polypeptides in equal amounts as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A cDNA for one of the polypeptides, now designated proPO-p2, has been isolated (Hall et al. (1995) Proc. Natl. Acad. Sci. USA, 92, 7764-7768). We purified pro-PO from plasma of M. sexta and characterized its subunit composition. A cDNA for M. sexta proPO-p1 was isolated from a larval hemocyte cDNA library. M. sexta proPO-p1 is 78% identical in amino acid sequence to Bombyx mori proPO-p1, but only 50% to M. sexta or B. mori proPO-p2. Immunofluorescence labelling and in situ hybridization showed that the pro-PO is synthesized in a single hemocyte type, the oenocytoids. Analysis of pro-PO by size exclusion high-pressure liquid chromatography (HPLC) revealed that pro-PO exists as monomeric, dimeric, trimeric or multimeric structures depending on the ionic strength. All of these isoforms of the protein have phenol oxidase activity upon activation with a detergent, cetylpyridinium chloride. In analysis by non-denaturing PAGE, the majority of the purified pro-PO was present as two dimers of distinct mobility (fast and slow forms). Both forms contain proPO-p1 and proPO-p2, suggesting that they are heterodimers. Individual larvae can contain the slow form, the fast form, or both, which suggests that the slow and fast forms of proPO are allelic variants. These results indicate that there are two pro-PO genes in M. sexta, which are coordinately expressed in oenocytoids, and whose products form predominantly heterodimers in plasma.     

Cell adhesion properties of hemolin, an insect immune protein in the Ig superfamily

The isolation of antibacterial peptides from the giant silkmoth Hyalophora cecropia has opened the area of animal antibiotics [Boman, H. G. (1991) Cell 65, 205-207] and the study of insect immune genes has revealed striking similarities to many immune response genes in mammals [Hultmark, D. (1994) Nature 267, 116-117]. However, the molecules and mechanisms behind primordial immune recognition are not understood. One candidate for one such recognition molecule is hemolin, a 48-kDa immunoglobulin-related protein first isolated from H. cecropia, where it is up-regulated upon infection and secreted into the hemolymph. Hemolin was shown to bind to bacteria and to hemocytes, giving rise to changes in hemocyte adhesiveness and intracellular phosphorylation patterns [Faye, I. & Kanost, M. (1997) in Molecular mechanisms of immune responses in insects (Brey, P. T. & Hultmark, D., eds) Chapman and Hall, London]. In the present publication, we give evidence for the presence of a 52-kDa membrane form of hemolin on hemocytes, based on flow-activated cell sorting and membrane protein extractions. In addition we reveal calcium-dependent homophilic binding properties of hemolin, using hemolin-coated microspheres. When biotinylated recombinant hemolin was allowed to bind to hemocyte membranes, higher molecular-mass complexes were formed. Furthermore, we used immunological methods and Northern-blot analysis to demonstrate the presence of hemolin in embryos and retinal discs, suggesting that hemolin is expressed in several tissues at different developmental stages. These results show novel cell adhesion features of hemolin, corroborating its multifunctional character with putative roles in cellular and humoral immunity and in development.     

A role for the Drosophila Toll/Cactus pathway in larval hematopoiesis

In the Drosophila larva, blood cells or hemocytes are formed in the lymph gland. The major blood cell type, called plasmatocyte, is small, non-adhesive and phagocytic. Plasmatocytes differentiate into adhesive lamellocytes to form multilayered capsules around foreign substances or, in mutant melanotic tumor strains, around self tissue. Mutations in cactus or Toll, or constitutive expression of dorsal can induce lamellocyte differentiation and cause the formation of melanotic capsules. As maternally encoded proteins, Toll, Cactus and Dorsal, along with Tube and Pelle, participate in a common signal transduction pathway to specify the embryonic dorsal-ventral axis. Using the maternal pathway as a paradigm, we investigated if these proteins have additional roles in larval hemocyte formation and differentiation. Analysis of cactus mutants that lack Cactus protein revealed that almost all of these animals have an overabundance of hemocytes, carry melanotic capsules and die before reaching pupal stages. In addition, the lymph glands of cactus larvae are considerably enlarged. The number of mitotic cells in the cactus and TollD hemolymph is higher than that in the wild-type hemolymph. The hemocyte density of mutant Toll, tube or pelle hemolymph is significantly lower than that of the wild type. Lethality of mutant cactus animals could be rescued either by the selective expression of wild-type Cactus protein in the larval lymph gland or by the introduction of mutations in Toll, tube or pelle. Cactus, Toll, Tube and Pelle proteins are expressed in the nascent hemocytes of the larval lymph gland. Our results suggest that the Toll/Cactus signal transduction pathway plays a significant role in regulating hemocyte proliferation and hemocyte density in the Drosophila larva. These findings are discussed in light of similar hematopoietic functions of Rel/I(kappa)B-family proteins in mice.     

Effects of midgut-protein-preparative and ligand binding procedures on the toxin binding characteristics of BT-R1, a common high-affinity receptor in Manduca sexta for Cry1A Bacillus thuringiensis toxins

The identity of the physiologically important Cry1A receptor protein(s) in the lepidopteran Manduca sexta has been a matter of dispute due to the multiple proteins which bind the Cry1Ac toxin. Cry1Aa, Cry1Ab, and Cry1Ac exhibit essentially identical toxicities toward M. sexta larvae and show a high degree of sequence and presumed structural identities. These similarities make it likely that there is a common mechanism of toxicity in these lepidopteran-specific toxins in terms of both mode of action and the receptor proteins through which these toxins exert their lepidopteran-specific toxicity. Investigators in our laboratory previously demonstrated that the cloned 210-kDa glycoprotein BT-R1 binds all three Cry1A toxins (T. P. Keeton and L. A. Bulla, Jr., Appl. Environ. Microbiol. 63:3419-3425, 1997). This protein remains a common binding protein even after being subjected to various midgut membrane preparation and processing protocols. The method used to isolate proteins from the M. sexta larval midgut in no significant way affects the results of ligand binding and vacuum blotting experiments, and we have been unable to detect specific, high-affinity binding of any Cry1A toxin to Cry1Ac binding proteins other than BT-R1. Alterations in blot substrate and blocking, hybridization, and washing buffers support these conclusions. Collectively, these results indicate that in M. sexta the cadherin-like BT-R1 protein is a common high-affinity receptor protein for the Cry1A family of toxins.     

Allatostatin-like-immunoreactive neurons of the tobacco hornworm, Manduca sexta, and isolation and identification of a new neuropeptide related to cockroach allatostatins

The YXFGLamide C-terminus serves to define most members of a family of structurally related neuropeptides, the YXFGLamides. These peptides have been identified from the nervous system of various insects and include the allatostatins of cockroaches and crickets, the schistostatins of locusts, and the callatostatins of blowflies. The YXFGLamides have been shown to have various functions, including inhibition of juvenile hormone biosynthesis in cockroaches and crickets and inhibition of contraction of certain insect visceral muscles. We wanted to know if these peptides occur in Manduca sexta and what functions they might have. A new peptide, AKSYNFGLamide, was isolated and identified from M. sexta and has been named "lepidostatin-1"; this is the first YXFGLamide to be found in a lepidopteran, and there are indications that additional YXFGLamides occur in M. sexta. An antiserum to cockroach allatostatins (YXFGLamides) was shown to recognize lepidostatin-1 of M. sexta and was used to map YXFGLamide-immunoreactive neurons in larvae. Because immunoreactive interneurons were found to form an extensive neuropil, YXFGLamides probably function as neuromodulators in M. sexta. Neuroendocrine cells in the brain, abdominal ganglia, and their respective neurohemal organs were YXFGLamide immunoreactive and appear to release YXFGLamides as neurohormones. Immunoreactivity to YXFGLamides and M. sexta diuretic hormone were found to be colocalized and appear to be coreleased in these neuroendocrine cells, indicating that YXFGLamides may be involved in regulation of fluid transport. Innervation of the corpora allata by YXFGLamide-immunoreactive processes was very sparse, suggesting that this innervation does not play an important role in allatostasis. Many thoracic motor neurons were YXFGLamide immunoreactive, suggesting that YXFGLamides may have a myomodulatory or myotrophic function in larvae. However, this immunoreactivity disappeared early in metamorphosis and did not reappear in the adult. The YXFGLamide-immunoreactive neurons in the terminal abdominal ganglion were found to innervate the hindgut, indicating that YXFGLamides may be involved in the control of the rate of myogenic contractions of the larval hindgut.     

Pro-phenol oxidase activating proteinase from an insect, Manduca sexta: a bacteria-inducible protein similar to Drosophila easter

Activation of pro-phenol oxidase (proPO) in insects and crustaceans is important in defense against wounding and infection. The proPO zymogen is activated by a specific proteolytic cleavage. PO oxidizes phenolic compounds to produce quinones, which may help to kill pathogens and can also be used for synthesis of melanin to seal wounds and encapsulate parasites. We have isolated from the tobacco hornworm, Manduca sexta, a serine proteinase that activates proPO, and have cloned its cDNA. The isolated proPO activating proteinase (PAP) hydrolyzed artificial substrates but required other protein factors for proPO activation, suggesting that proPO-activating enzyme may exist as a protein complex, one component of which is PAP. PAP (44 kDa) is composed of two disulfide-linked polypeptide chains (31 kDa and 13 kDa). A cDNA for PAP was isolated from a hemocyte library, by using a PCR-generated probe based on the amino-terminal amino acid sequence of the 31-kDa catalytic domain. PAP belongs to a family of arthropod serine proteinases containing a carboxyl-terminal proteinase domain and an amino-terminal "clip" domain. The member of this family most similar in sequence to PAP is the product of the easter gene from Drosophila melanogaster. PAP mRNA was present at a low level in larval hemocytes and fat body, but became much more abundant in fat body after insects were injected with Escherichia coli. Sequence data and 3H-diisopropyl fluorphosphate labeling results suggest that the same PAP exists in hemolymph and cuticle.     

Expression and hemocyte-targeting of a Campoletis sonorensis polydnavirus cysteine-rich gene in Heliothis virescens larvae

The polydnavirus associated with the parasitic wasp Campoletis sonorensis is injected into the lepidopteran insect, Heliothis virescens, during parasitization, after which viral gene products suppress the cellular immune system of the hosts. Four related cysteine-rich polydnavirus gene have been identified in parasitized H. virescens larvae and grouped into a family. In this study, we investigated the expression and hemocyte targeting of the cysteine-rich VHv1.4 protein. Full-length and truncated VHv1.4 proteins were produced in a bacterial expression system, and the purified proteins were used to raise polyclonal antisera. In immunoblots the VHv1.4 protein was detected in parasitized insects as early as 6 h and throughout the entire course of parasitism. The VHv1.4 protein appeared predominantly in the plasma fraction of hemolymph from parasitized larvae, suggesting that this protein is secreted. The VHv1.4 protein expressed from a recombinant baculovirus was secreted in two lepidopteran cell lines and in larvae injected with the recombinant virus. Digestion with endoglycosidases suggests that the VHv1.4 protein is glycosylated at multiple N-glycosylation sites. Immunofluorescence assays showed that the VHv1.4 protein binds to the hemocytes, most notably the granulocytes, in H. virescens larvae. After binding, the VHv1.4 protein was internalized, probably by endocytosis. Specific binding of the VHv1.4 to granulocytes implies an important function in the suppression of host cellular encapsulation response.     

The use of decapitated insects to study lipid mobilization in adult Manduca sexta: effects of adipokinetic hormone and trehalose on fat body lipase activity

In order to perform studies on lipid mobilization in adult M. sexta, it is necessary to overcome the effects of starvation and handling, which both provoke an increase in hemolymph lipid concentration. When trehalose was injected into intact insects, a 35% decrease in the content of the diacylglycerol (DG)-rich hemolymph lipoprotein, low density lipophorin (LDLp) was observed within 30 min, but the level of LDLp returned to control values after 1 h. Decapitated insects exhibited 60% reduction in LDLp concentration and the levels remained low for at least 24 h. In contrast to intact insects, injection of trehalose into decapitated animals did not alter the LDLp concentration. After decapitation, the response to adipokinetic hormone (AKH) and the ability of the fat body to release DG into the hemolymph was maintained for at least 24 h. In decapitated insects, 6 pmol of AKH-stimulated measurable lipid mobilization and a near maximum response was obtained with 100 pmol of the hormone. The action of trehalose and AKH on the fat body triacylglycerol (TG)-lipase activity in decapitated animals was studied. Fat body homogenates from trehalose-treated insects exhibited a TG-lipase activity 40% lower than the control insects. Activation of fat body triacylglycerol-lipase was observed after injection of AKH, with the extent of activation ranging between 97 and 380% ten min after AKH injection. A time course study showed that the activation of the fat body triacylglycerol lipase preceded the increase in hemolymph LDLp concentration, suggesting that activation of the lipase initiates lipid mobilization. It is concluded that decapitated insects injected with trehalose is a very useful system for investigating the hormonal regulation of lipid mobilization in adult M. sexta.     

A peptidyl-prolyl cis/trans-isomerase (cyclophilin G) in regulated secretory granules

A 27-kDa protein (p27) in horseshoe crab hemocyte that cross-reacts with antiserum against a beta-glucan-sensitive protease zymogen was purified to homogeneity, and its cDNA was cloned. The 1.7-kilobase pair cDNA contains an open reading frame of 660 base pairs, encoding a 23-amino acid signal sequence followed by a mature protein of 197 residues. The sequence of p27 exhibits strong similarity to that of cyclophilin B, a peptidyl-prolyl cis/trans-isomerase. p27 exhibits isomerase activity with a kcat/Km of 0.18 microM-1 s-1 for a peptide substrate; this activity is inhibited by cyclosporin A but is not affected by FK506. Although the p27 precursor possesses an amino-terminal secretory hydrophobic signal sequence, unlike other cyclophilin B molecules, it lacks a conserved carboxyl-terminal endoplasmic reticulum retention signal and it contains a central 8-amino acid insertion. Although p27 is secreted into the culture media of transiently expressed COS cells, it is not detected in horseshoe crab hemolymph plasma but rather is localized to the hemocyte large granules, the regulated secretory granules that are exocytosed upon stimulation. These results indicate that p27 is a new peptidyl-prolyl cis/trans-isomerase in the regulated secretory granules, and is thus designated cyclophilin G. This first report of a cyclophilin homologue in the secretory granule of the horseshoe crab hemocyte suggests that such chaperon-like proteins may constitute a key quality control system for stored proteins in exocytotic granules.     

The development and utility of a defined muscle and fat co-culture system

Various subspecies of the gram-positive bacterium Bacillus thuringiensis are known to produce a wide array of insecticidal crystal proteins (ICPs) upon sporulation. These ICPs act primarily on the brush border of midgut epithelial cells of susceptible larvae. Recently, a protein of 210 kDa, isolated from the midgut of Manduca sexta, has been demonstrated to bind the Cry1Ab toxin produced by B. thuringiensis subsp, berliner and is therefore postulated to be involved in mediating the toxicity of Cry1Ab. The cDNA encoding the 210 kDa protein, termed BT-R1 (Bacillus thuringiensis receptor-1), was recently cloned, and shows limited homology to the cadherin superfamily of proteins. Quite naturally, there is a great deal of interest in the characterization of BT-R1, the gene encoding the 210 kDa Cry1Ab binding protein. The studies presented here involve the use of various restriction fragments prepared from the cDNA encoding BT-R1 as probes of Southern blots bearing M. sexta genomic DNA cleaved with a variety of restriction endonucleases. These Southern blot data reveal that there are two discrete regions within the M. sexta genome which encode sequences homologous to BT-R1. On the basis of the signal intensities seen on Southern blots, it appears that only one of these genes encodes BT-R1, whereas the other is a closely related homologue.     

Expression of superoxide dismutase following axotomy

Genomic clones for a chitinolytic enzyme were isolated from a library of Sau 3A digested DNA from the tobacco hornworm, Manduca sexta, using a previously isolated chitinase cDNA clone as a probe [Kramer et al., Insect Biochem. Molec. Biol. 23, 691-701 (1993)]. Restriction enzyme mapping and Southern blot analysis of four genomic clones suggested that these are overlapping clones. Sequence analysis of the genomic clones and Southern blot analysis of total genomic DNA also suggest that the M. sexta genome has only one chitinase gene detectable by the cDNA probe. This gene is organized into at least 11 exons in a region spanning > 11 kb. The sequenced M. sexta chitinase gene has a series of exons corresponding to identifiable structural/functional regions of the protein. Similarities in structure and organization between the M. sexta chitinase gene and chitinase genes from other sources are described.     

Tachycitin, a small granular component in horseshoe crab hemocytes, is an antimicrobial protein with chitin-binding activity

Small granules of horseshoe crab hemocytes contain two known major antimicrobial substances, tachyplesin and big defensin (S5), and at least five protein components (S1 to S6), with unknown functions. In the present study, we examined the biological properties and primary structure of a small granular component S2, named tachycitin. This component was purified from the acid extract of hemocyte debris by two steps of chromatography. The purified tachycitin was a single chain protein with an apparent M(r) = 8,500 on Tricine-SDS-polyacrylamide gel electrophoresis. Ultracentrifugation analysis revealed tachycitin to be present in monomer form in solution. Tachycitin inhibited the growth of both Gram-negative and -positive bacteria, and fungi, with a bacterial agglutinating property. Moreover, tachycitin and big defensin acted synergistically in antimicrobial activities. The amino acid sequence and intrachain disulfide bonds of tachycitin were determined by amino acid and sequence analyses of peptides produced by enzymatic cleavages. The mature tachycitin consisted of 73 amino acid residues containing five disulfide bonds with no N-linked sugar. A cDNA coding for tachycitin was isolated from a hemocyte cDNA library. The open reading frame coded for an NH2-terminal signal sequence followed by the mature peptide and an extension sequence of -Gly-Arg-Lys at the COOH-terminus, which is a putative amidating signal. The COOH-terminal threonine amide released after digestion of tachycitin with lysylendopeptidase was identified. The NH2-terminal 28 residues of tachycitin shows sequence homology to a part of chitin-binding regions found in antifungal chitin-binding peptides, chitin-binding lectins, and chitinases, all of which have been isolated from plants. Tachycitin showed a specific binding to chitin but did not bind with the polysaccharides cellulose, mannan, xylan, and laminarin. Tachycitin may represent a new class of chitin-binding protein family in animals.     

Disruption of lysosomal targeting is associated with insecticidal potency of juvenile hormone esterase

Juvenile hormone esterase (JHE; EC 3.1.1.1), which is intrinsically involved in regulation of development of some insect larvae, is rapidly removed from the hemolymph by the pericardial cells. Lys-29 and Lys-524, which are implicated in the degradation of JHE, were mutated to Arg. Neither the half-life of the modified JHE in the hemolymph nor the catalytic parameters were changed significantly, but when combined, these mutations resulted in apparent failure of lysosomal targeting in the pericardial cell complex. A hypothesis for the mechanism of reduced efficiency of lysosomal targeting is presented. Infection of larvae with a recombinant baculovirus expressing the modified JHE resulted in a 50% reduction in feeding damage compared with larvae infected with the wild-type virus, thus demonstrating improved properties as a biological insecticide. These data demonstrate that alteration of specific residues of JHE that disrupted lysosomal targeting, dramatically increased the insecticidal activity of this protein.     

Recombinant human interleukin-1-mediated killing of Schistosoma mansoni primary sporocysts in Biomphalaria glabrata

Previous work has indicated that injection of recombinant-human interleukin (rhIL)-1beta in Schistosoma mansoni-infected M-line Biomphalaria glabrata resulted in a significant reduction in the number of cercariae shed. The purpose of the present work was to determine if primary sporocysts were killed following rhIL-1beta injection in susceptible snails and, if so, to determine if killing was the direct result of hemocyte activity. Counting of primary sporocysts indicated a 50% reduction in the number surviving at 3 days PE in snails from 2 susceptible strains following injection. Histological analysis indicated that killing occurred with little-to-no observable hemocyte/parasite contact, whereas short-term culture of primary sporocysts with cell-free plasma (hemolymph) from injected snails rapidly initiated killing in vitro. Because levels of a snail IL-1-like molecule (SnaIL-1) drop significantly following schistosome exposure in M-line snails, because resistant snails maintain higher SnaIL-1 levels following infection, and because rhIL-1beta upregulates hemocyte cytotoxic mechanisms, these data support the contention that SnaIL-1 plays a role in determining resistance in B. glabrata. These data also indicate that schistosome death may be separated from parasite encapsulation by hemocytes and that an as yet unidentified humoral killing mechanism/factor may exist in B. glabrata. Lastly, these data further support the hypothesis that cytokine-like molecules are important, functionally conserved immunodefense mediators in both vertebrates and invertebrates.     

Protein kinase C is activated in platelets subjected to pathological shear stress

High levels of fluid shear stress at the blood vessel wall directly stimulate von Willebrand factor (vWF)-mediated platelet adhesion and aggregation and thereby contribute to the pathogenesis of arterial thrombosis. We have found that a pathological level of arterial wall shear stress (90 dynes/cm2) induces platelet aggregation that is associated with the phosphorylation of pleckstrin, a M(r) 47,000 protein kinase C substrate (p47). Shear-induced p47 phosphorylation depends entirely on vWF binding to platelet glycoprotein (Gp) Ib and GpIIb-IIIa, and the specific inhibition of protein kinase C with the staurosporine analogue Ro 31-7549 inhibits the full aggregation response to shear. Shear stress-induced platelet p47 phosphorylation occurs independent of any measurable change in diacylglycerol mass or hydrolysis of phosphatidylinositol 4,5-bisphosphate. These results indicate that mechanical shear stress induces vWF to bind to platelet GpIb and GpIIb-IIIa, stimulating a diacylglycerol-independent pathway of protein kinase C activation that contributes to platelet aggregation in response to shear.     

Absence of short-term regulation over gluconeogenesis by glucose in the insect Manduca sexta L

In vivo gluconeogenesis from (3-13 C)alanine was evident in terminal instar Manduca sexta larvae from the selective fractional 13C enrichment in trehalose, a disaccharide of glucose and the major blood sugar of insects. De novo glucose synthesis was observed in insects fed a low carbohydrate diet for 1 or more days. Gluconeogenesis was not inhibited by a single injection of glucose nor by short-term feeding on glucose-supplemented diet. Reduced fractional 13C enrichment in trehalose was demonstrated upon glucose administration, but was explained by isotopic dilution following direct synthesis of trehalose from the unlabeled glucose. Isotopic dilution was also quantified by analysis of the 13C labeling pattern in trehalose synthesized following injection of (1,2-13C2)glucose. The results suggest the absence of short-term regulation over gluconeogenesis by glucose and may partially explain why blood sugar level in M. sexta and other insects fluctuates over a wide concentration range. Although glucose had no observable effects on gluconeogenesis, injection of or feeding glucose resulted in a significantly increased activity of the pentose phosphate pathway.     

Identification and characterization of a novel beta subunit of soluble guanylyl cyclase that is active in the absence of a second subunit and is relatively insensitive to nitric oxide

Previously characterized soluble guanylyl cyclases form alpha-beta heterodimers that can be activated by the gaseous messenger, nitric oxide. In mammals, four subunits have been cloned, named alpha1, alpha2, beta1, and beta2. We have identified a novel soluble guanylyl cyclase isoform from the nervous system of the insect Manduca sexta that we have named M. sexta guanylyl cyclase beta3 (MsGC-beta3). It is most closely related to the mammalian beta subunits but has several features that distinguish it from previously identified soluble cyclases. Most importantly, MsGC-beta3 does not need to form heterodimers to form an active enzyme because guanylyl cyclase activity can be measured when it is expressed alone in COS-7 cells. Moreover, this activity is only weakly enhanced in the presence of the nitric oxide donor, sodium nitroprusside. Several of the amino acids in rat beta1 subunits, previously identified as being important in heme binding or necessary for nitric oxide activation, are substituted with nonsimilar amino acids in MsGC-beta3. There are also an additional 315 amino acids C-terminal to the catalytic domain of MsGC-beta3 that have no sequence similarity to any known protein. Northern blot analysis shows that MsGC-beta3 is primarily expressed in the nervous system of Manduca.     

Protoporphyrinogen oxidase of Myxococcus xanthus. Expression, purification, and characterization of the cloned enzyme

Protoporphyrinogen oxidase (EC 1.3.3.4) catalyzes the six electron oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from the bacterium Myxococcus xanthus has been cloned, expressed, purified, and characterized. The protein has been expressed in Escherichia coli using a Tac promoter-driven expression plasmid and purified to apparent homogeneity in a rapid procedure that yields approximately 10 mg of purified protein per liter of culture. Based upon the deduced amino acid sequence the molecular weight of a single subunit is 49,387. Gel permeation chromatography in the presence of 0.2% n-octyl-beta-D-glucopyranoside yields a molecular weight of approximately 100,000 while SDS gel electrophoresis shows a single band at 50,000. The native enzyme is, thus, a homodimer. The purified protein contains a non-covalently bound FAD but no detectable redox active metal. The M. xanthus enzyme utilizes protoporphyrinogen IX, but not coproporphyrinogen III, as substrate and produces 3 mol of H2O2/mol of protoporphyrin. The apparent Km and kcat for protoporphyrinogen in assays under atmospheric concentrations of oxygen are 1.6 microM and 5.2 min-1, respectively. The diphenyl ether herbicide acifluorfen at 1 microM strongly inhibits the enzyme's activity.