The enhanced in vitro hematopoietic activity of leridistim, a chimeric dual G-CSF and IL-3 receptor agonist.

The in vitro activity of leridistim was characterized for cell proliferation, generation of colony-forming units (CFU) and differentiation of CD34+ cells. In AML-193.1.3 cells, leridistim exhibited a significant increase in potency compared to rhG-CSF, SC-65303 (an IL-3 receptor agonist) or an equimolar combination of rhG-CSF and SC-65303. CFU-GM assays demonstrated that at 50% of the maximum response, the relative potency of leridistim was 12-fold greater than the combination of rhG-CSF and rhIL-3 and 44-fold more potent than rhG-CSF alone. In multi-lineage CFU assays, a combination of erythropoietin (rhEPO) and leridistim resulted in greater numbers of BFU-E, CFU-GEMM and CFU-Mk than rhEPO alone. Ex vivo culture of peripheral blood or bone marrow CD34+ cells with leridistim substantially increased total viable cells over cultures stimulated with rhG-CSF, SC-65303, or a combination of rhG-CSF and SC-65303. Culture with leridistim, resulted in a greater increase in myeloid (CD15+/CD11b+), monocytic (CD41-/CD14+) and megakaryocytic (CD41+/CD14-) precursor cells without depleting the progenitor pool (CD34+/CD15-/CD11b-). These results demonstrate that leridistim is a more potent stimulator of hematopoietic proliferation and differentiation than the single receptor agonists (rhG-CSF and SC-65303) either alone or combined. These unique attributes suggest that leridistim may enhance hematopoietic reconstitution following myelosuppressive chemotherapy.

Transfer of sulfur from IscS to IscU during Fe/S cluster assembly.

The cysteine desulfurase enzymes NifS and IscS provide sulfur for the biosynthesis of Fe/S proteins. NifU and IscU have been proposed to serve as template or scaffold proteins in the initial Fe/S cluster assembly events, but the mechanism of sulfur transfer from NifS or IscS to NifU or IscU has not been elucidated. We have employed [(35)S]cysteine radiotracer studies to monitor sulfur transfer between IscS and IscU from Escherichia coli and have used direct binding measurements to investigate interactions between the proteins. IscS catalyzed transfer of (35)S from [(35)S]cysteine to IscU in the absence of additional thiol reagents, suggesting that transfer can occur directly and without involvement of an intermediate carrier. Surface plasmon resonance studies and isothermal titration calorimetry measurements further revealed that IscU binds to IscS with high affinity (K(d) approximately 2 microm) in support of a direct transfer mechanism. Transfer was inhibited by treatment of IscU with iodoacetamide, and (35)S was released by reducing reagents, suggesting that transfer of persulfide sulfur occurs to cysteinyl groups of IscU. A deletion mutant of IscS lacking C-terminal residues 376-413 (IscSDelta376-413) displayed cysteine desulfurase activity similar to the full-length protein but exhibited lower binding affinity for IscU, decreased ability to transfer (35)S to IscU, and reduced activity in assays of Fe/S cluster assembly on IscU. The findings with IscSDelta376-413 provide additional support for a mechanism of sulfur transfer involving a direct interaction between IscS and IscU and suggest that the C-terminal region of IscS may be important for binding IscU.

Progenipoietins: biological characterization of a family of dual agonists of fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor.

The progenipoietins, a class of engineered proteins containing both fetal liver tyrosine kinase-3 and granulocyte colony-stimulating factor receptor agonist activities, were functionally characterized in vitro and in vivo. Four representative progenipoietins were evaluated for receptor binding, receptor-dependent cell proliferation, colony-forming unit activity, and their effects on hematopoiesis in the C57BL/6 mouse.The progenipoietins bound to fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor with affinities within twofold to threefold of the native ligands, and each progenipoietin bound simultaneously to both fetal liver tyrosine kinase-3 and the granulocyte colony-stimulating factor receptor. The progenipoietins exhibited different levels of activity in receptor-dependent cell proliferation assays. The fetal liver tyrosine kinase-3-dependent cell proliferation activity of three of four progenipoietins was decreased sixfold to 33-fold relative to native fetal liver tyrosine kinase-3 ligand, while granulocyte colony-stimulating factor receptor-dependent activity of the progenipoietins was within twofold to threefold of native granulocyte colony-stimulating factor. At nonsaturating concentrations, the progenipoietins stimulated colony formation to a greater extent than the equimolar combination of fetal liver tyrosine kinase-3 and granulocyte colony-stimulating factor. Treatment of mice with the progenipoietins yielded dramatic increases in peripheral blood and splenic white blood cells, polymorphonuclear leukocytes, and dendritic cells. These preclinical results demonstrate that the progenipoietins are potent hematopoietic growth factors that stimulate cells in a receptor-dependent manner. When administered in vivo, the progenipoietins effectively promote the generation of multiple cell lineages. Thus, in both in vitro and in vivo settings, the progenipoietins as single molecules exhibit the synergistic activity of the combination of fetal liver tyrosine kinase-3 and granulocyte colony-stimulating factor.

The Fe/S assembly protein IscU behaves as a substrate for the molecular chaperone Hsc66 from Escherichia coli.

IscU, a NifU-like Fe/S-escort protein, binds to and stimulates the ATPase activity of Hsc66, a hsp70-type molecular chaperone. We present evidence that stimulation arises from interactions of IscU with the substrate-binding site of Hsc66. IscU inhibited the ability of Hsc66 to suppress the aggregation of the denatured model substrate proteins rhodanese and citrate synthase, and calorimetric and surface plasmon resonance measurements showed that ATP destabilizes Hsc66.IscU complexes in a manner expected for hsp70-substrate complexes. Studies on the interaction of IscU with Hsc66 truncation mutants further showed that IscU does not bind the isolated ATPase domain of Hsc66 but does bind and stimulate a mutant containing the ATPase domain and substrate binding beta-sandwich subdomain. These results support a role for IscU as a substrate for Hsc66 and suggest a specialized function for Hsc66 in the assembly, stabilization, or transfer of Fe/S clusters formed on IscU.

Crystal structure of Hsc20, a J-type Co-chaperone from Escherichia coli.

Hsc20 is a 20 kDa J-protein that regulates the ATPase activity and peptide-binding specificity of Hsc66, an hsp70-class molecular chaperone. We report herein the crystal structure of Hsc20 from Escherichia coli determined to a resolution of 1.8 A using a combination of single isomorphous replacement (SIR) and multi-wavelength anomalous diffraction (MAD). The overall structure of Hsc20 consists of two distinct domains, an N-terminal J-domain containing residues 1-75 connected by a short loop to a C-terminal domain containing residues 84-171. The structure of the J-domain, involved in interactions with Hsc66, resembles the alpha-topology of J-domain fragments of Escherichia coli DnaJ and human Hdj1 previously determined by solution NMR methods. The C-terminal domain, implicated in binding and targeting proteins to Hsc66, consists of a three-helix bundle in which two helices comprise an anti-parallel coiled-coil. The two domains make contact through an extensive hydrophobic interface ( approximately 650 A(2)) suggesting that their relative orientations are fixed. Thus, Hsc20, in addition to its role in the regulation of the ATPase activity of Hsc66, may also function as a rigid scaffold to facilitate positioning of the protein substrates targeted to Hsc66.

Interaction of the iron-sulfur cluster assembly protein IscU with the Hsc66/Hsc20 molecular chaperone system of Escherichia coli.

The iscU gene in bacteria is located in a gene cluster encoding proteins implicated in iron-sulfur cluster assembly and an hsc70-type (heat shock cognate) molecular chaperone system, iscSUA-hscBA. To investigate possible interactions between these systems, we have overproduced and purified the IscU protein from Escherichia coli and have studied its interactions with the hscA and hscB gene products Hsc66 and Hsc20. IscU and its iron-sulfur complex (IscU-Fe/S) stimulated the basal steady-state ATPase activity of Hsc66 weakly in the absence of Hsc20 but, in the presence of Hsc20, increased the ATPase activity up to 480-fold. Hsc20 also decreased the apparent K(m) for IscU stimulation of Hsc66 ATPase activity, and surface plasmon resonance studies revealed that Hsc20 enhances binding of IscU to Hsc66. Surface plasmon resonance and isothermal titration calorimetry further showed that IscU and Hsc20 form a complex, and Hsc20 may thereby aid in the targeting of IscU to Hsc66. These results establish a direct and specific role for the Hsc66/Hsc20 chaperone system in functioning with isc gene components for the assembly of iron-sulfur cluster proteins.

Kinetic characterization of the ATPase cycle of the molecular chaperone Hsc66 from Escherichia coli.

Hsc66 from Escherichia coli is a constitutively expressed hsp70 class molecular chaperone whose activity is coupled to ATP binding and hydrolysis. To better understand the mechanism and regulation of Hsc66, we investigated the kinetics of ATP hydrolysis and the interactions of Hsc66 with nucleotides. Steady-state experiments revealed that Hsc66 has a low affinity for ATP (K(m)(ATP) = 12.7 microM) compared with other hsp70 chaperones. The kinetics of nucleotide binding were determined by analyzing changes in the Hsc66 absorbance spectrum using stopped-flow methods at 23 degrees C. ATP binding results in a rapid, biphasic increase of Hsc66 absorbance at 280 nm; this is interpreted as arising from a two-step process in which ATP binding (k(a)(ATP) = 4.2 x 10(4) M(-1) s(-1), k(d)(ATP) = 1.1 s(-1)) is followed by a slow conformational change (k(conf) = 0. 1 s(-1)). Under single turnover conditions, the ATP-induced transition decays exponentially with a rate (k(decay) = 0.0013 s(-1)) similar to that observed in both steady-state and single turnover ATP hydrolysis experiments (k(hyd) = 0.0014 s(-1)). ADP binding to Hsc66 results in a monophasic transition in the absence (k(a)(ADP) = 7 x 10(5) M(-1) s(-1), k(d)(ADP) = 60 s(-1)) and presence of physiological levels of inorganic phosphate (k(a)(ADP(P(i)) = 0.28 x 10(5) M(-1) s(-1), k(d)(ADP(P(i)) = 9.1 s(-1)). These results indicate that ATP hydrolysis is the rate-limiting step under steady-state conditions and is >10(3)-fold slower than the rate of ADP/ATP exchange. Thus, in contrast to DnaK and eukaryotic forms of hsp70 that have been characterized to date, the R if T equilibrium balance for Hsc66 is shifted in favor of the low peptide affinity T state, and regulation of the reaction cycle is expected to occur at the ATP hydrolysis step rather than at nucleotide exchange.

Saccharomyces cerevisiae ISU1 and ISU2: members of a well-conserved gene family for iron-sulfur cluster assembly.

Recent studies in bacteria and eukaryotes have led to the identification of several new genes implicated in the biogenesis of iron-sulfur (Fe/S) cluster-containing proteins. This report focuses on two genes of bakers yeast Saccharomyces cerevisiae, ISU1 and ISU2, which encode homologues to bacterial IscU and NifU, potential iron-binding or cluster-assembly proteins. As with other yeast genes implicated in Fe/S protein assembly, deletion of either ISU1 or ISU2 results in increased accumulation of iron within the mitochondria, loss of activity of the [4Fe-4S] aconitase enzyme, and suppression of oxidative damage in cells lacking cytosolic copper/zinc superoxide dismutase. Both genes are induced in strains expressing an activated allele of Aft1p, the iron-sensing transcription factor, suggesting that they are regulated by the iron status of the cell. Immunoblotting studies using an antibody directed against Escherichia coli IscU reveal that both Isu1p and Isu2p are localized primarily in the mitochondria and that Isu1p is the predominant form expressed under all growth conditions tested. The possible role of the Isu proteins in the assembly and/or repair of Fe/S clusters is discussed.

The Hsc66-Hsc20 chaperone system in Escherichia coli: chaperone activity and interactions with the DnaK-DnaJ-grpE system.

Hsc66, a stress-70 protein, and Hsc20, a J-type accessory protein, comprise a newly described Hsp70-type chaperone system in addition to DnaK-DnaJ-GrpE in Escherichia coli. Because endogenous substrates for the Hsc66-Hsc20 system have not yet been identified, we investigated chaperone-like activities of Hsc66 and Hsc20 by their ability to suppress aggregation of denatured model substrate proteins, such as rhodanese, citrate synthase, and luciferase. Hsc66 suppressed aggregation of rhodanese and citrate synthase, and ATP caused effects consistent with complex destabilization typical of other Hsp70-type chaperones. Differences in the activities of Hsc66 and DnaK, however, suggest that these chaperones have dissimilar substrate specificity profiles. Hsc20, unlike DnaJ, did not exhibit intrinsic chaperone activity and appears to function solely as a regulatory cochaperone protein for Hsc66. Possible interactions between the Hsc66-Hsc20 and DnaK-DnaJ-GrpE chaperone systems were also investigated by measuring the effects of cochaperone proteins on Hsp70 ATPase activities. The nucleotide exchange factor GrpE did not stimulate the ATPase activity of Hsc66 and thus appears to function specifically with DnaK. Cross-stimulation by the cochaperones Hsc20 and DnaJ was observed, but the requirement for supraphysiological concentrations makes it unlikely that these interactions occur significantly in vivo. Together these results suggest that Hsc66-Hsc20 and DnaK-DnaJ-GrpE comprise separate molecular chaperone systems with distinct, nonoverlapping cellular functions.

Suppressors of superoxide dismutase (SOD1) deficiency in Saccharomyces cerevisiae. Identification of proteins predicted to mediate iron-sulfur cluster assembly.

Yeast deficient in the cytosolic copper/zinc superoxide dismutase (SOD1) exhibit metabolic defects indicative of oxidative damage even under non-stress conditions. To help identify the endogenous sources of this oxidative damage, we isolated mutant strains of S. cerevisiae that suppressed metabolic defects associated with loss of SOD1. Six complementation groups were isolated and three of the corresponding genes have been identified. One sod1Delta suppressor represents SSQ1 which encodes a hsp70-type molecular chaperone found in the mitochondria. A second sod1Delta suppressor gene, designated JAC1, represents a new member of the 20-kDa J-protein family of co-chaperones. Jac1p contains a mitochondrial targeting consensus sequence and may serve as the partner for Ssq1p. Homologues of Ssq1p and Jac1p are found in bacteria in close association with genes proposed to be involved in iron-sulfur protein biosynthesis. The third suppressor gene identified was NFS1. Nfs1p is homologous to cysteine desulfurase enzymes that function in iron-sulfur cluster assembly and is also predicted to be mitochondrial. Each of the suppressor mutants identified exhibited diminished rates of respiratory oxygen consumption and was found to have reduced mitochondrial aconitase and succinate dehydrogenase activities. Taken together these results suggest a role for Ssq1p, Jac1p, and Nfs1p in assembly/maturation of mitochondrial iron-sulfur proteins and that one or more of the target Fe/S proteins contribute to oxidative damage in cells lacking copper/zinc SOD.

Crystallization and preliminary X-ray crystallographic properties of Hsc20, a J-motif co-chaperone protein from Escherichia coli.

Hsc20 is a 20-kDa auxiliary protein that functions with the molecular chaperone Hsc66 in Escherichia coli. Crystals of Hsc20 suitable for X-ray diffraction analysis were grown using the hanging drop vapor diffusion technique in polyethylene glycol 400 containing dioxane as an additive to slow growth. The crystals are monoclinic and belong to the space group C2 with unit cell dimensions a = 125.4 A, b = 71.9 A, c = 68.8 A, and beta = 97.0 degrees. The crystals diffract to a minimum d-spacing of approximately 2.5 A resolution, and a native data set was collected to 2.7 A. The results of a self-rotation function analysis revealed threefold symmetry, suggesting three molecules of Hsc20 in the asymmetric unit and, hence, 12 molecules in the unit cell; this corresponds to a Vm value of 2.6 A3/Da and a solvent content of approximately 53% in the crystals. Structure determination by isomorphous replacement is in progress.

Hsc66 and Hsc20, a new heat shock cognate molecular chaperone system from Escherichia coli.

The hscA and hscB genes of Escherichia coli encode novel chaperone and co-chaperone proteins, designated Hsc66 and Hsc20, respectively. We have overproduced and purified Hsc66 and Hsc20 in high yield in E. coli and describe their initial characterization including absorbance, fluorescence, and circular dichroism spectra. Immunoblot analyses of E. coli cultures using antisera to Hsc66 and Hsc20 raised in rabbits establish that Hsc66 and Hsc20 are constitutively expressed at levels corresponding to cell concentration approximately 20 microM and approximately 10 microM, respectively. The levels do not change appreciably following heat shock (44 degrees C), but a small increase in Hsc20 is observed following a shift to 10 degrees C. Purified Hsc66 exhibits a low intrinsic ATPase activity (approximately 0.6 min-1 at 37 degrees C), and Hsc20 was found to stimulate this activity up to 3.8-fold with half-maximal stimulation at a concentration approximately 5 microM. These findings suggest that Hsc66 and Hsc20 comprise a molecular chaperone system similar to the prokaryotic DnaK/DnaJ and eukaryotic hsp70/hsp40 systems. Sequence differences between Hsc66 and Hsc20 compared to other members of this chaperone family, however, suggest that the Hsc66/Hsc20 system will display different peptide binding specificity and that it is likely to be subject to different regulatory mechanisms. The high level of constitutive expression and the lack of a major response to temperature changes suggest that Hsc66 and Hsc20 play an important cellular role(s) under non-stress conditions.

Cooperativity and dimerization of recombinant human estrogen receptor hormone-binding domain.

The estrogen receptor dimerizes and exhibits cooperative ligand binding as part of its normal functioning. Interaction of the estrogen receptor with its ligands is mediated by a C-terminal hormone-binding domain (HBD), and residues within the HBD are thought to contribute to dimerization. To examine dimer interactions in the isolated HBD, a human estrogen receptor HBD fragment was expressed in high yield as a cleavable fusion protein in Escherichia coli. The isolated HBD peptide exhibited affinity for estradiol, ligand discrimination, and cooperative estradiol binding (Hill coefficient approximately 1.6) similar to the full-length protein. Circular dichroism spectroscopy suggests that the HBD contains significant amounts of alpha-helix ( approximately 60%) and some beta-strand ( approximately 7%) and that ligand binding induces little change in secondary structure. HBD dimer dissociation, measured using size exclusion chromatography, exhibited a half-life of approximately 1.2 h, which ligand binding increased approximately 3-fold (estradiol) to approximately 4-fold (4-hydroxytamoxifen). These results suggest that the isolated estrogen receptor HBD dimerizes and undergoes conformational changes associated with cooperative ligand binding in a manner comparable to the full-length protein, and that one effect of ligand binding is to alter the receptor dimer dissociation kinetics.

Molecular recognition and electron transfer in mitochondrial steroid hydroxylase systems.

Mitochondrial monooxygenase systems are involved in the biosynthesis of glucocorticoids, mineralocorticoids, bile acids, and 1,25-dihydroxyvitamin D. The reactions are catalyzed by specific P450 enzymes that receive reducing equivalents via NADPH-ferredoxin oxidoreductase (adrenodoxin reductase) and ferredoxin (adrenodoxin). Although the three-dimensional structures of the individual components have not yet been solved, methods of expressing recombinant forms of these enzymes in Escherichia coli have allowed the use of site-directed mutagenesis to investigate the roles of specific amino acids in protein binding interactions, electron transfer, and catalysis. These studies have identified key charged residues in NADPH-ferredoxin oxidoreductase, ferredoxin, and P450scc, which are involved in electrostatic interactions critical for recognition, high-affinity binding, and electron transfer. The finding that the binding sites on ferredoxin for NADPH-ferredoxin oxidoreductase and P450 show significant overlap supports the proposed function for ferredoxin as a mobile electron shuttle between the reductase and P450 enzymes and is consistent with ferredoxin's role in serving multiple P450 isoforms.

Multinuclear magnetic resonance and mutagenesis studies of the histidine residues of human mitochondrial ferredoxin.

Human mitochondrial ferredoxin is a [2Fe-2S] protein that functions to transfer electrons from NADPH-dependent ferredoxin reductase to cytochrome P450 enzymes. Two of the three histidines of human ferredoxin are strictly conserved in the sequences of all known vertebrate ferredoxins, and one of these (His56) is adjacent to Cys55, which serves as one of the ligands to the iron-sulfur cluster. All but 16 of its residues show sequence identity with those of bovine ferredoxin. It has been proposed for bovine ferredoxin that His56 hydrogen bonds with a labile sulfur and that the reduction of the iron-sulfur center is accompanied by the uptake of a proton by this histidine [Lambeth, J. D., Seybert, D. W., Lancaster, J. R., Jr., Salerno, J. C., & Kamin, H. (1982) Mol. Cell. Biochem. 45, 13-31]. In this paper, we report procedures for labeling human ferredoxin uniformly with 15N using 15NH4Cl and selectively with 13C by the incorporation of [U-13C]histidine. Most of the imidazole 1H, 13C, and 15N resonances of the three histidines have been assigned by heteronuclear two-dimensional single- and multiple-bond correlation spectroscopy. Site-directed mutagenesis was used in assigning the NMR signals from His56. The pKa values of His10 (6.5) and His62 (5.8) in oxidized human ferredoxin were found to be similar to those reported previously for the corresponding residues of bovine ferredoxin [Greenfield, N. J., Wu, X., & Jordan, F. (1989) Biochim. Biophys. Acta 995, 246-254; Miura, S., Tamita, S., & Ichikawa, Y. (1991) J. Biol. Chem. 266, 19212-19216].(ABSTRACT TRUNCATED AT 250 WORDS)

A gene encoding a DnaK/hsp70 homolog in Escherichia coli.

Eukaryotic organisms have been shown to have multiple forms of hsp70-class stress-related proteins, but only a single family member, DnaK, has been found in prokaryotes. We report here the identification of a heat shock cognate gene, designated hsc, in Escherichia coli. The amino acid sequence deduced from hsc predicts a 65,647-Da polypeptide having 41% sequence identity with DnaK from E. coli, and overexpression produces a protein (Hsc66) with properties similar to DnaK. In contrast to dnaK, however, the hsc gene lacks a consensus heat shock promoter sequence, and expression is not induced by elevated temperature. The hsc gene is located near 54 min on the physical map, immediately upstream of the fdx gene, which encodes a [2Fe-2S] ferredoxin; evidence is presented that the hsc and fdx genes make up a bicistronic operon in which expression of the ferredoxin is coupled to that of Hsc66. The function of Hsc66 is not known, but the coregulation of its expression with that of ferredoxin suggests the possibility of a specific role in association with the ferredoxin protein.

Charge pair interactions stabilizing ferredoxin-ferredoxin reductase complexes. Identification by complementary site-specific mutations.

Ferredoxin reductase (Fd-reductase) supplies electrons to mitochondrial steroid hydroxylase cytochrome P450 enzymes via a [2Fe-2S] ferredoxin. Chemical labeling studies with bovine Fd-reductase have implicated Lys-243 as important in binding to bovine ferredoxin (Hamamoto, I., Kazutaka, K., Tanaka, S., and Ichikawa, Y. (1988) Biochim. Biophys. Acta 953, 207-213). We have used site-directed mutagenesis to examine the role of charged residues in this region of human Fd-reductase in ferredoxin binding. Mutant proteins were expressed in Escherichia coli and were assayed for activity by ferredoxin-mediated electron transfer to cytochrome c. Replacement of Lys-242 (homologous to Lys-243 in bovine Fd-reductase) with Gln and replacement of Arg-241 with Ser had little effect (2.7- and 3.6-fold increased Km, respectively). In contrast, mutants at positions 239 and 243 (R239S and R243Q) exhibited markedly lower affinity for ferredoxin (17.5- and 1,600-fold increased Km, respectively). Studies were also carried out with two ferredoxin charge mutants shown previously to have lowered affinity for Fd-reductase (Coghlan, V. M., and Vickery, L. E. (1991) J. Biol. Chem. 266, 18606-18612). Comparisons of the binding of ferredoxin mutants D76N and D79N to Fd-reductase mutants R239S and R243Q suggest that Arg-239 and Arg-243 of Fd-reductase each interact directly with both Asp-76 and Asp-79 of ferredoxin during formation of the complex between the two proteins. These results support the hypothesis that specific electrostatic interactions involving this region are important in stabilizing the ferredoxin-Fd-reductase complex.

Inhibition of human aromatase by mammalian lignans and isoflavonoid phytoestrogens.

Isoflavonoid phytoestrogens and lignans in plants are known to be constituents of animal and human food and recently they have been found in human urine and other biological materials. These compounds have received increasing attention because of their interesting biological properties and possible role in human cancer and other diseases. The present study demonstrates that the main mammalian lignan enterolactone (trans-2,3-bis[(3-hydroxyphenyl)methyl]-butyrolactone) and some other diphenols are moderate or weak inhibitors of human estrogen synthetase (aromatase) and that this lignan binds to or near the substrate region of the active site of the P-450 enzyme. The inhibition is competitive with respect to testosterone and androstenedione, and the lignan affinity is 1/75-1/300 that of these natural substrates. It is suggested that the high concentration of lignans in vegetarians, by inhibiting aromatase in peripheral and/or cancer cells and lowering estrogen levels, may play a protective role as antipromotional compounds during growth of estrogen-dependent cancers.

Cloning, sequencing, and overexpression of a [2Fe-2S] ferredoxin gene from Escherichia coli.

Escherichia coli contains a soluble, [2Fe-2S] ferredoxin of unknown function (Knoell, H.-E., and Knappe, J. (1974) Eur. J. Biochem. 50, 245-252). Using antiserum to the purified protein to screen E. coli genomic expression libraries, we have cloned a gene (designated fdx) encoding this protein. The DNA sequence of the gene predicts a polypeptide of 110 residues after removal of the initiator methionine (polypeptide M(r) = 12,186, holoprotein M(r) = 12,358). The deduced amino acid sequence is strikingly similar to those of the ferredoxins found in animal mitochondria which function with cytochrome P450 enzymes and to the ferredoxin from Pseudomonas putida which functions with P450cam. The overall sequence identity is approximately 36% when compared with human mitochondrial and P. putida ferredoxins, and the identities include 4 cysteine residues proposed to coordinate the iron cluster. The protein was overproduced approximately 500-fold using an expression plasmid, and the holoprotein was assembled and accumulated in amounts exceeding 30% of the total cell protein. The overexpressed ferredoxin exhibits absorption, circular dichroism, and electron paramagnetic resonance spectra closely resembling those of the animal ferredoxins and P. putida ferredoxin.