Bacterioferritin: a key iron storage modulator that affects strain growth and butenyl-spinosyn biosynthesis in Saccharopolyspora pogona.

BACKGROUND: Butenyl-spinosyn, produced by Saccharopolyspora pogona, is a promising biopesticide due to excellent insecticidal activity and broad pesticidal spectrum. Bacterioferritin (Bfr, encoded by bfr) regulates the storage and utilization of iron, which is essential for the growth and metabolism of microorganisms. However, the effect of Bfr on the growth and butenyl-spinosyn biosynthesis in S. pogona has not been explored. RESULTS: Here, we found that the storage of intracellular iron influenced butenyl-spinosyn biosynthesis and the stress resistance of S. pogona, which was regulated by Bfr. The overexpression of bfr increased the production of butenyl-spinosyn by 3.14-fold and enhanced the tolerance of S. pogona to iron toxicity and oxidative damage, while the knockout of bfr had the opposite effects. Based on the quantitative proteomics analysis and experimental verification, the inner mechanism of these phenomena was explored. Overexpression of bfr enhanced the iron storage capacity of the strain, which activated polyketide synthase genes and enhanced the supply of acyl-CoA precursors to improve butenyl-spinosyn biosynthesis. In addition, it induced the oxidative stress response to improve the stress resistance of S. pogona. CONCLUSION: Our work reveals the role of Bfr in increasing the yield of butenyl-spinosyn and enhancing the stress resistance of S. pogona, and provides insights into its enhancement on secondary metabolism, which provides a reference for optimizing the production of secondary metabolites in actinomycetes.

Crucial role of two potential cytosolic regions of Nox2, 191TSSTKTIRRS200 and 484DESQANHFAVHHDEEKD500, on NADPH oxidase activation.

Assembly of cytosolic factors p67(phox) and p47(phox) with cytochrome b(558) is one of the crucial keys for NADPH oxidase activation. Certain sequences of Nox2 appear to be involved in cytosolic factor interaction. The role of the D-loop (191)TSSTKTIRRS(200) and the C-terminal (484)DESQANHFAVHHDEEKD(500) of Nox2 on oxidase activity and assembly was investigated. Charged amino acids were mutated to neutral or reverse charge by directed mutagenesis to generate 21 mutants. Recombinant wild-type or mutant Nox2 were expressed in the X-CGD PLB-985 cell model. K195A/E, R198E, R199E, and RR198199QQ/AA mutations in the D-loop of Nox2 totally abolished oxidase activity. However, these D-loop mutants demonstrated normal p47(phox) translocation and iodonitrotetrazolium (INT) reductase activity, suggesting that charged amino acids of this region are essential for electron transfer from FAD to oxygen. Replacement of Nox2 D-loop with its homolog of Nox1, Nox3, or Nox4 was fully functional. In addition, fMLP (formylmethionylleucylphenylalanine)-activated R199Q-Nox2 and D-loop(Nox4)-Nox2 mutants exhibited four to eight times the NADPH oxidase activity of control cells, suggesting that these mutations lead to a more efficient oxidase activation process. In contrast, the D484T and D500A/R/G mutants of the alpha-helical loop of Nox2 exhibited no NADPH oxidase and INT reductase activities associated with a defective p47(phox) membrane translocation. This suggests that the alpha-helical loop of the C-terminal of Nox2 is probably involved in the correct assembly of the NADPH oxidase complex occurring during activation, permitting cytosolic factor translocation and electron transfer from NADPH to FAD.

Synthetic peptides corresponding to various hydrophilic regions of the large subunit of cytochrome b558 inhibit superoxide generation in a cell-free system from neutrophils.

Cytochrome b558 is a component of the superoxide-generating system in neutrophils and plays key roles in both the assembly of a functional complex with cytosolic proteins and shuttling an electron from NADPH to molecular oxygen. To determine the role of predicted hydrophilic domains of gp91-phox, a glycosylated subunit of the cytochrome, we synthesized peptides corresponding to the regions and tested whether they affected superoxide generation in the cell-free system obtained from human neutrophils. Among twelve peptides tested, six peptides, four of which correspond to previously unreported regions, inhibited superoxide generation in the cell-free system. All of the active peptides were effective when added to the system before activation with sodium dodecyl sulfate. Four peptides, including two peptides corresponding to two newly identified regions, inhibited the translocation of the cytosolic components, p47-phox and p67-phox. The extent of inhibition on translocation of these components varied depending on the peptide used.

Characterization of peptide diffusion into electropermeabilized neutrophils.

The superoxide (O2-)-generating NADPH oxidase of human neutrophils consists of membrane-bound and cytosolic proteins that assemble in the plasma membrane of activated cells. To date, most of our understanding of the assembly of the NADPH oxidase has been obtained through the use of a cell-free assay, and a number of peptides that mimic regions of NADPH oxidase proteins have been shown to block oxidase assembly using this assay. However, the cell-free assay provides an incomplete representation of the assembly and regulation of the NADPH oxidase in vivo, and it has become necessary to develop methods for introducing biomolecules, such as peptides, into intact neutrophils where their effects can be investigated. One such method is electropermeabilization. Although this method has been used previously with human neutrophils, it has not been well characterized. We report here a detailed characterization of the electropermeabilized neutrophil assay system, including optimal conditions for membrane electropermeabilization with maximal retention of functional capacity, optimal conditions for analyzing the effects of experimental peptides, quantification of internalized peptide concentration, and molecular size limits for diffusion of molecules into these cells. Our results demonstrate that optimal neutrophil permeabilization (98-100%) can be achieved using significantly lower electrical fields than previously reported, resulting in the retention of higher levels of O2(-)-generating activity. We also found that biomolecules as large as 2.3 kDa readily diffuse into permeabilized cells. Analysis of flavocytochrome b peptides that were shown previously to inhibit NADPH oxidase activity in a cell-free assay demonstrated that these peptides also blocked O2- production in electropermeabilized human neutrophils; although at higher effective concentrations than in the cell-free system. Thus, electropermeabilized neutrophils provide a model system for evaluating the effects of peptides and other pharmacological agents in intact cells which closely mimic neutrophils in vivo.

Characterization of superoxide dismutase-insensitive cytochrome c reductase activity in HL-60 cytosol as NADPH-cytochrome P450 reductase.

A flavoprotein dehydrogenase assayed for the activity of electron transfer from NADPH to cytochrome c was highly purified from the cytosolic fraction of differentiated human promyelocytic leukemia HL-60 cells. The purified enzyme had an apparent molecular mass of 68 kDa by sodium dodecyl sulfate gel electrophoresis and an equimolar amounts of flavin mononucleotide and flavin-adenine dinucleotide. The purification factor of the enzyme with respect to the cytosolic fraction was close to 1100 and the recovery of activity was approximately 18%. Reduction of cytochrome c by NADPH indicated Michaelis-Menten kinetics with a Km value of 1.50 microM for NADPH. When cytochrome c was the varied substrate, a Km value of 4.10 microM was obtained. NADH was not an effective electron donor for cytochrome c reduction and NADPH-dependent reduction of nitroblue tetrazolium was negligibly small. The purified enzyme alone did not exhibit superoxide production, and NADPH oxidase activity was not markedly stimulated upon incubation of the reductase with cytochrome b558 purified from porcine neutrophils. The purified flavoprotein gave a positive cross-reactivity to polyclonal antibodies raised to microsomal NADPH-cytochrome P450 reductase, indicating structural homology between these enzymes. The catalytic properties of the purified NADPH-cytochrome c reductase have similarities to those of liver NADPH-cytochrome P450 reductase.

Ethanol-inducible cytochrome P450 in rabbits metabolizes enflurane.

Following anaesthesia with enflurane, some patients receiving isoniazid have increased serum concentrations of fluoride ion, presumably because of induction of an isozyme of cytochrome P450 which is responsible for enflurane biodegradation. In rats, isoniazid and ethanol enhance metabolism of enflurane and also induce a form of cytochrome P450 which is homologous with a form of rabbit liver cytochrome P450 known as 3a. Isoniazid, ethanol and imidazole increase the concentration of cytochrome P450 3a in hepatic microsomes. We have pretreated rabbits with imidazole, the most potent of the three inducers of isozyme 3a, to determine if the hepatic microsomal metabolism of enflurane is enhanced and if purified isozyme 3a catalyses the oxidation of enflurane. Imidazole produced a 250% increase in the hepatic microsomal metabolism of enflurane, sevoflurane, methoxyflurane and the control substrate, aniline. Polyclonal antibodies to cytochrome P450 3a inhibited 90% of enflurane metabolism, but only 40% of methoxyflurane biotransformation in the microsomes from imidazole-pretreated rabbits. Thus isozyme 3a or a structurally similar cytochrome P450 seemed to catalyse almost all microsomal metabolism of enflurane. In addition, purified cytochrome P450 3a catalysed the metabolism of enflurane, sevoflurane and methoxyflurane, and the oxidation of these anaesthetics by cytochrome P450 3a was stimulated four-fold by cytochrome b5, a protein which serves as an alternate source of electrons for some cytochrome P450 reactions.

Phosphorylation of cytochrome P450: regulation by cytochrome b5.

Rabbit liver cytochrome P450 LM2 and several forms of rat liver cytochrome P450 are phosphorylated by cAMP-dependent protein kinase (PKA) and by protein kinase C. Under aqueous assay conditions at neutral pH LM2 is phosphorylated only to a maximum extent of about 20 mol% by PKA. We show that detergents or alkaline pH greatly enhance the extent of phosphorylation of the cytochrome P450 substrates of cAMP-dependent protein kinase. In the presence of 0.05% Emulgen, PBRLM5, which appears to be the best cytochrome P450 substrate for cAMP-dependent protein kinase, incorporates phosphate up to about 84 mol% of enzyme. We reported previously (I. Jansson et al. (1987) Arch. Biochem. Biophys. 259, 441-448) that cytochrome b5 inhibits the phosphorylation of LM2 by cAMP-dependent protein kinase. In this paper, using PBRLM5, we demonstrate, by analysis of initial rates, that the inhibition of phosphorylation by cytochrome b5 is competitive, with a Ki = 0.48 microM. We also show that a number of forms of cytochrome P450 can be phosphorylated by protein kinase C, and that the phosphorylation of these forms by protein kinase C is also inhibited by cytochrome b5. These data suggest that the phosphorylation site(s) of cytochromes P450 may be located within or overlap the cytochrome b5 binding domain of the enzymes.

Substrate specificity and alkyl group selectivity in the metabolism of N-nitrosodialkylamines.

Metabolic activation may be a key step in determining the tissue specificity of carcinogenic nitrosamines. In previous work, we characterized P450IIE1 (an acetone/ethanol-inducible form of cytochrome P-450) as the major enzyme for the metabolic activation of N-nitrosodimethylamine. In this work, we investigated the metabolism of other N-nitrosodialkylamines in rat liver microsomes and in reconstituted monooxygenase systems containing purified cytochrome P-450 isozymes. The enzyme specificities in the metabolism of N-nitrosoethylmethylamine and N-nitrosodiethylamine were similar to those of N-nitrosodimethylamine; i.e., these substrates were more efficiently metabolized by acetone- or ethanol-induced microsomes than by other types of microsomes. However, substituting one methyl group with a benzyl or butyl group, as in N-nitrosobenzylmethylamine or N-nitrosobutylmethylamine (NBMA), substantially changed the enzyme specificity. P450IIE1 efficiently catalyzed the demethylation but not the debutylation of NBMA, whereas P450IIB1 (a phenobarbital-inducible form) efficiently catalyzed both the debutylation and demethylation reactions. In the demethylation of NBMA by P450IIE1, the addition of cytochrome b5 markedly increased the activity at low but not at high substrate concentrations, suggesting a decrease in Km value. This effect, however, was not observed in the debutylation of NBMA by P450IIE1 or P450IIB1, and in the demethylation of NBMA by P450IIB1. These studies demonstrate the substrate specificity and alkyl group selectivity in the metabolism of nitrosamines by cytochrome P-450 isozymes.

Constitutive testosterone 6 beta-hydroxylase in rat liver.

The cytochrome P-450 that was purified from hepatic microsomes of male rats treated with phenobarbital and designated P450 PB-1 (Funae and Imaoka (1985) Biochim. Biophys. Acta 842, 119-132) had high testosterone 6 beta-hydroxylation activity (turnover rate, 13.5 nmol of product/min/nmol of P-450) in a reconstituted system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5, and a 1:1 mixture of lecithin and phosphatidylserine in the presence of sodium cholate. In ordinary conditions in the reconstituted system with cytochrome P-450, reductase, and dilauroylphosphatidylcholine, P450 PB-1 had little 6 beta-hydroxylase activity. The catalytic activities toward testosterone of two major constitutive forms, P450 UT-2 and P450 UT-5, were not affected by cytochrome b5, phospholipid, or sodium cholate. P450 PB-1 in rat liver microsomes was assayed by immunoblotting with specific antibody to P450 PB-1. P450 PB-1 accounted for 24.4 +/- 5.6% (mean +/- SD) of the total spectrally-measured cytochrome P-450 in hepatic microsomes of untreated adult male rats, and was not found in untreated adult female rats. P450 PB-1 was induced twofold with phenobarbital in male rats. P450 PB-1 was purified from untreated male rats and identified as P450 PB-1 from phenobarbital-treated rats by its NH2-terminal sequence, peptide mapping, and immunochemistry. These results showed that P450 PB-1 is a constitutive male-specific form in rat liver. There was a good correlation (r = 0.925) between the P450 PB-1 level and testosterone 6 beta-hydroxylase activity in rat liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Reductive metabolism of halothane by purified cytochrome P-450.

The reductive metabolism of halothane was determined using purified RLM2, PBRLM4 and PBRLM5 forms of rat liver microsomal cytochrome P-450. The metabolites, 2-chloro-1,1,1-trifluoroethane (CTE) and 2-chloro-1,1-difluoroethylene (CDE), were determined. All three forms of cytochrome P-450 produced CTE with relatively small differences in its production among the various forms. There were major differences, however, in the production of CDE, with PBRLM5 being the most active. PBRLM5 was also the only form to show the development of a complex between halothane and cytochrome P-450. This complex absorbed light maximally at 470 nm. The complex formation and the production of CDE by PBRLM5 were stimulated by the addition of cytochrome b5. Cytochrome b5 had no effect on CDE production by PBRLM4 and inhibited the production of both CTE and CDE by RLM2. These results show that the two-electron reduction of halothane by cytochrome P-450 was catalyzed by the PBRLM5 form and that cytochrome b5 stimulated the transfer of the second electron to halothane through PBRLM5, but not RLM2 or PBRLM4.

Influence of cytochrome b5 on the stoichiometry of the different oxidative reactions catalyzed by liver microsomal cytochrome P-450.

Stoichiometries of oxygen and NADPH consumption and product and hydrogen peroxide formation are examined for three forms of cytochrome P-450, LM2, RLM2, and RLM5, using several different substrates. As reported earlier, during the metabolism of some substrates [Gorsky, Koop, and Coon: J. Biol. Chem. 259, 6812-6817 (1984)], excess NADPH and oxygen are consumed suggesting that a 4-electron reduction of oxygen to water occurs. Similar effects are seen with testosterone as substrate for the constitutive forms of P-450, RLM2 and RLM5. However, when aminopyrine or p-nitroanisole serve as substrate, none of the forms of P-450 consumed excess NADPH or oxygen. Thus, consumption of excess NADPH and oxygen appears to be the result of the substrate used. Cytochrome b5 stimulates turnover of LM2 and RLM5 but not RLM2. With LM2, it causes a metabolic switching to occur between the monooxygenase and the NADPH-oxidase reaction. Although RLM5 is also stimulated by cytochrome b5, no metabolic switching occurs. Cytochrome b5 did not affect the proportion of excess NADPH or oxygen consumed.

Incorporation of cytochrome b5 into rat liver microsomal membranes. Impairment of cytochrome P-450-dependent mixed function oxidase activity.

Cytochrome b5 was purified to electrophoretic homogeneity from the liver microsomes of untreated rats and reincorporated into liver microsomes from phenobarbital-treated rats, resulting in an approximate three-fold enrichment of the cytochrome b5 specific content (1.5 nmol haemoprotein X mg-1 protein). Our results have shown that the N-demethylation of benzphetamine was progressively inhibited in cytochrome b5-fortified microsomal preparations. Using stopped flow, visible difference spectrophotometry, the NADPH-driven reduction kinetics of cytochrome P-450 were examined in the modified microsomes over the first few seconds of reaction. Increasing the amount of incorporated cytochrome b5 resulted in a progressive inhibition of the initial, fast phase reduction rate constant of microsomal cytochrome P-450, both in the absence and presence of the type I substrate benzphetamine. Although the initial rate of NADPH-driven cytochrome b5 reduction was the same for both native and cytochrome b5-fortified microsomes, the extent of cytochrome b5 reduction was greater in the fortified microsomes. If cytochrome b5 has a positive role to play in cytochrome P-450-dependent mixed function oxidase activity either as an effector or in electron transfer or both, the former haemoprotein must be already present in sufficient concentrations in the native microsomes.

N-demethylation of N-nitrosodimethylamine catalyzed by purified rat hepatic microsomal cytochrome P-450: isozyme specificity and role of cytochrome b5.

Metabolism of the potent hepatocarcinogen N-nitrosodimethylamine (NDMA) was evaluated in reconstituted monooxygenase systems containing each of 11 purified rat hepatic cytochrome P-450 isozymes. The reaction has an absolute requirement for cytochrome P-450, NADPH-cytochrome P-450 reductase, and NADPH, as well as a partial dependence on dilauroylphosphatidylcholine. Of the cytochrome P-450 isozymes evaluated, only cytochrome P-450j, purified from livers of ethanol- or isoniazid-treated rats, had high catalytic activity for the N-demethylation of NDMA. At substrate concentrations of 0.5 and 5 mM, rates of NDMA metabolism to formaldehyde catalyzed by cytochrome P-450j were at least 15-fold greater than the rates obtained with any of the other purified isozymes. At the pH optimum (approximately 6.7) for the reaction, the Km,app and Vmax were 3.5 mM and 23.9 nmol/min/nmol cytochrome P-450j, respectively. With hepatic microsomes from ethanol-treated rats, which contain induced levels of cytochrome P-450j, the Km,app and Vmax were 0.35 mM and 3.9 nmol/min/nmol cytochrome P-450, respectively. Inclusion of purified cytochrome b5 in the reconstituted system containing cytochrome P-450j caused a six-fold decrease in Km,app (0.56 mM) of NDMA demethylation with little or no change in Vmax (29.9 nmol/min/nmol cytochrome P-450j). Trypsin-solubilized cytochrome b5, bovine serum albumin, or hemoglobin had no effect on the kinetic parameters of the reconstituted system, indicating a specific effect of intact cytochrome b5 on the Km,app of the reaction. These results demonstrate high isozyme specificity in the metabolism of NDMA to an ultimate carcinogen and further suggest an important role for cytochrome b5 in this biotransformation process.

Effects of cytochrome b5 on aniline hydroxylation catalyzed by a reconstituted system containing acetone or 2,2'-bipyridine.

Cytochrome b5 did not exert any effect on NADPH-dependent aniline hydroxylation in the absence of acetone or 2,2'-bipyridine, whereas cytochrome b5 exhibited a stimulatory effect on the reaction in the presence of acetone or 2,2'-bipyridine. In addition, cytochrome b5 did not have any significant effect on the cumene hydroperoxide-dependent reaction in the presence of acetone or 2,2'-bipyridine.

Effects of conditions for reconstitution with cytochrome b5 on the formation of products in cytochrome P-450-catalyzed reactions.

Evidence is presented that the method of reconstitution of the cytochrome P-450-containing liver microsomal enzyme system with cytochrome b5 (b5), including the order of addition of the components, the concentration of the b5, and the length of incubation prior to initiation of the reaction by NADPH, governs the steady state catalytic activity obtained. For example, the addition to cytochrome P-450 isozyme 2, NADPH-cytochrome P-450 reductase, and phosphatidylcholine of concentrated b5 (0.4 microM) results in extensive inhibition of benzphetamine demethylation and NADPH oxidation, whereas the addition of dilute b5 (0.02 microM) to the other components results in extensive stimulation of the demethylation reaction. The inhibition is partly relieved by prolonged incubation. The effects of pH and buffer concentration were determined, and the optimal molar ratio of b5 to cytochrome P-450 isozyme 2 was shown to be about 2.0 for stimulation of benzphetamine demethylation, dimethylaniline demethylation, and cyclohexanol oxidation to cyclohexanone. Cytochrome P-450 isozyme 4-catalyzed aminopyrine demethylation and aniline p-hydroxylation are not stimulated by b5, as predicted from a model based on stopped flow kinetic measurements [Pompon and Coon: J. Biol. Chem. 259, 15377 (1984)]. End-point stoichiometry measurements were carried out with cytochrome P-450 isozyme 2 in the absence of b5 or in the presence of b5 under optimal conditions. The results indicate that when b5 is reconstituted with the cytochrome P-450 isozyme 2 enzyme system under optimal conditions, substrate monooxygenation is enhanced, NADPH oxidation is unaffected, and hydrogen peroxide formation is decreased.

Isolation by ion-exchange high performance liquid chromatography of rabbit liver cytochrome P-450 with regioselectivity for omega-hydroxylation of prostaglandins.

Previous studies suggested that rabbit liver microsomes contain cytochrome P-450 monooxygenase(s) with low affinity for (omega-1)-hydroxylation and high affinity for omega-hydroxylation of prostaglandins (Theoharides, A. D., and Kupfer, D. (1981) J. Biol. Chem. 256, 2168-2175). The current investigation describes the isolation from livers of untreated rabbits of a cytochrome P-450 catalyzing, with regioselectivity, the omega-hydroxylation of prostaglandins E1 and E2. The isolation of the enzyme involved enrichment of the omega-hydroxylase activity by polyethylene glycol 8000 fractionation, followed by ion-exchange high performance liquid chromatography. Based on Mr of 59,000-60,000 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the isolated enzyme is referred to as P-450 form 7. This P-450 exhibits a low spin spectrum (lambda max = 417 nm) and a difference spectrum of the CO-reduced complex versus reduced (lambda max = 451 nm). For catalytic activity, the P-450 form 7 was reconstituted with NADPH-P-450 reductase, cytochrome b5, and lipid. There was no activity in the absence of the reductase, and deletion of cytochrome b5 yielded a minimal amount of product (heme could not substitute for cytochrome b5), demonstrating an absolute requirement for these components.

Conversion of oxyhaemoglobin into methaemoglobin by ferricytochrome b5.

Ferricytochrome b5 was found to convert oxyhaemoglobin into methaemoglobin under conditions previously found to be optimal for complex-formation between ferricytochrome b5 and methaemoglobin [Mauk & Mauk (1982) Biochemistry 21, 4730-4734]. As this reaction is completely inhibited by CO, it is proposed that oxyhaemoglobin is oxidized after O2 dissociation, as has been suggested for the oxidation of oxyhaemoglobin by inorganic complexes. From the present analysis, ferricytochrome b5 seems unlikely to contribute significantly to methaemoglobin formation in vivo. Nevertheless, this observation provides a relatively convenient means of investigating the mechanism by which these two proteins interact.