An Enzyme-Mediated Aza-Michael Addition Is Involved in the Biosynthesis of an Imidazoyl Hybrid Product of Conidiogenone B.

Meleagrin B is a terpene-alkaloid hybrid natural product that contains both the conidiogenone and meleagrin scaffold. Their derivatives show diverse biological activities. We characterized the biosynthesis of (-)-conidiogenone B (1), which involves a diterpene synthase and a P450 monooxygenase. In addition, an α,ß-hydrolase (Con-ABH) was shown to catalyze an aza-Michael addition between 1 and imidazole to give 3S-imidazolyl conidiogenone B (6). Compound 6 was more potent than 1 against Staphylococcus aureus strains.

Unveiling sequential late-stage methyltransferase reactions in the meleagrin/oxaline biosynthetic pathway.

Antimicrobial and anti-proliferative meleagrin and oxaline are roquefortine C-derived alkaloids produced by fungi of the genus Penicillium. Tandem O-methylations complete the biosynthesis of oxaline from glandicoline B through meleagrin. Currently, little is known about the role of these methylation patterns in the bioactivity profile of meleagrin and oxaline. To establish the structural and mechanistic basis of methylation in these pathways, crystal structures were determined for two late-stage methyltransferases in the oxaline and meleagrin gene clusters from Penicillium oxalicum and Penicillium chrysogenum. The homologous enzymes OxaG and RoqN were shown to catalyze penultimate hydroxylamine O-methylation to generate meleagrin in vitro. Crystal structures of these enzymes in the presence of methyl donor S-adenosylmethionine revealed an open active site, which lacks an apparent base indicating that catalysis is driven by proximity effects. OxaC was shown to methylate meleagrin to form oxaline in vitro, the terminal pathway product. Crystal structures of OxaC in a pseudo-Michaelis complex containing sinefungin and meleagrin, and in a product complex containing S-adenosyl-homocysteine and oxaline, reveal key active site residues with His313 serving as a base that is activated by Glu369. These data provide structural insights into the enzymatic methylation of these alkaloids that include a rare hydroxylamine oxygen acceptor, and can be used to guide future efforts towards selective derivatization and structural diversification and establishing the role of methylation in bioactivity.

A natural short pathway synthesizes roquefortine C but not meleagrin in three different Penicillium roqueforti strains.

The production of mycotoxins and other secondary metabolites in Penicillium roqueforti is of great interest because of its long history of use in blue-veined cheese manufacture. In this article, we report the cloning and characterization of the roquefortine gene cluster in three different P. roqueforti strains isolated from blue cheese in the USA (the type strain), France, and the UK (Cheshire cheese). All three strains showed an identical roquefortine gene cluster organization and almost identical (98-99%) gene nucleotide sequences in the entire 16.6-kb cluster region. When compared with the Penicillium chrysogenum roquefortine/meleagrin seven-gene cluster, the P. roqueforti roquefortine cluster contains only four genes (rds, rdh, rpt, and gmt) encoding the roquefortine dipeptide synthetase, roquefortine D dehydrogenase, roquefortine prenyltransferase, and a methyltransferase, respectively. Silencing of the rds or rpt genes by the RNAi strategy reduced roquefortine C production by 50% confirming the involvement of these two key genes in roquefortine biosynthesis. An additional putative gene, orthologous of the MFS transporter roqT, is rearranged in all three strains as a pseudogene. The same four genes and a complete (not rearranged) roqT, encoding a MFS transporter containing 12 TMS domains, occur in the seven-gene cluster in P. chrysogenum although organized differently. Interestingly, the two "late" genes of the P. chrysogenum roquefortine/meleagrin gene cluster that convert roquefortine C to glandicoline B and meleagrin are absent in the P. roqueforti four-gene cluster. No meleagrin production was detected in P. roqueforti cultures grown in YES medium, while P. chrysogenum produces meleagrin in these conditions. No orthologous genes of the two missing meleagrin synthesizing genes were found elsewhere in the recently released P. roqueforti genome. Our data suggest that during evolution, the seven-gene cluster present in P. chrysogenum, and probably also in other glandicoline/meleagrin producing fungi, has been trimmed down to a short cluster in P. roqueforti leading to the synthesis of roquefortine C rather than meleagrin as a final product.

A branched biosynthetic pathway is involved in production of roquefortine and related compounds in Penicillium chrysogenum.

Profiling and structural elucidation of secondary metabolites produced by the filamentous fungus Penicillium chrysogenum and derived deletion strains were used to identify the various metabolites and enzymatic steps belonging to the roquefortine/meleagrin pathway. Major abundant metabolites of this pathway were identified as histidyltryptophanyldiketopiperazine (HTD), dehydrohistidyltryptophanyldi-ketopiperazine (DHTD), roquefortine D, roquefortine C, glandicoline A, glandicoline B and meleagrin. Specific genes could be assigned to each enzymatic reaction step. The nonribosomal peptide synthetase RoqA accepts L-histidine and L-tryptophan as substrates leading to the production of the diketopiperazine HTD. DHTD, previously suggested to be a degradation product of roquefortine C, was found to be derived from HTD involving the cytochrome P450 oxidoreductase RoqR. The dimethylallyltryptophan synthetase RoqD prenylates both HTD and DHTD yielding directly the products roquefortine D and roquefortine C without the synthesis of a previously suggested intermediate and the involvement of RoqM. This leads to a branch in the otherwise linear pathway. Roquefortine C is subsequently converted into glandicoline B with glandicoline A as intermediates, involving two monooxygenases (RoqM and RoqO) which were mixed up in an earlier attempt to elucidate the biosynthetic pathway. Eventually, meleagrin is produced from glandicoline B involving a methyltransferase (RoqN). It is concluded that roquefortine C and meleagrin are derived from a branched biosynthetic pathway.

A single cluster of coregulated genes encodes the biosynthesis of the mycotoxins roquefortine C and meleagrin in Penicillium chrysogenum.

A single gene cluster of Penicillium chrysogenum contains genes involved in the biosynthesis and secretion of the mycotoxins roquefortine C and meleagrin. Five of these genes have been silenced by RNAi. Pc21g15480 (rds) encodes a nonribosomal cyclodipeptide synthetase for the biosynthesis of both roquefortine C and meleagrin. Pc21g15430 (rpt) encodes a prenyltransferase also required for the biosynthesis of both mycotoxins. Silencing of Pc21g15460 or Pc21g15470 led to a decrease in roquefortine C and meleagrin, whereas silencing of the methyltransferase gene (Pc21g15440; gmt) resulted in accumulation of glandicolin B, indicating that this enzyme catalyzes the conversion of glandicolin B to meleagrin. All these genes are transcriptionally coregulated. Our results prove that roquefortine C and meleagrin derive from a single pathway.

Synthesis of the allergen ovomucoid by a replicating Mengo virus.

Interferons induced by viral infections can have powerful immuno- modulatory effects, and several epidemiologic studies have found an association between certain viral infections and reduced prevalence of allergy. We hypothesized that allergenic proteins could be synthesized by a replicating virus, and this construct could be useful as an immunomodulator. To test this hypothesis, we cloned an allergenic protein (ovomucoid [OVM]) into a murine picornavirus (Mengo virus) vector. This plasmid has a multicloning site surrounded by auto-catalytic sequences so that a foreign protein will be cleaved from viral proteins during replication. OVM sequences were cloned in the context of full-length viral genome cDNA, T7 RNA transcripts of this plasmid were transfected into HeLa cells, and recombinant virus plaques appeared on the second passage. Sequence analysis of recombinant viruses derived from individual plaques demonstrated that three viral isolates contained up to 2/3 of the OVM coding sequence, which was retained by the viruses after 5 additional passages in HeLa cells. The experiments verify the stable expression of immunoreactive OVM subunits by replicating viruses. These virus/allergen constructs could provide a tool to evaluate whether intracellular presentation of allergenic proteins in the context of a viral infection could prevent allergic sensitization upon re-challenge.

Structural and immunological characterization of recombinant ovomucoid expressed in Escherichia coli.

The expression of recombinant allergens is becoming new insights of an important diagnosis and the therapy of allergies as well as molecular approaches to immunological and structural studies of allergens. Ovomucoid is a major food allergens in the hen's egg white which causes immediate food-hypersensitivity reactions mainly in children. A gene coding for the cDNA representing an entire ovomucoid molecule has been cloned in Escherichia coli under the control of T5 promoter fused with six-Histidine tag at the amino terminal end. Upon induction, the E. coli cells, harbouring this construct, expressed the recombinant protein as a soluble fraction and the recombinant ovomucoid protein was purified to electrophoeretic homogeneity using Ni2+ nitrilotriacetic acid agarose affinity chromatography. Immunoblot analysis showed that human IgE and IgG binding activities of the recombinant ovomucoid was identical to that of native analogue. The antigenicity and allergenicity of recombinant ovomucoid were almost same as that of native form when tested with an ELISA using six individual patient's serum. CD spectra indicated that that the recombinant ovomucoid has more alpha-helix and less beta-structure than native form. These results show that the recombinant ovomucoid constructed in this study could be used for further studies on the immunological and structural studies of ovomucoid.

[The biosynthesis of low-molecular-weight nitrogen-containing secondary metabolite-alkaloids by the resident strains of Penicillium chrysogenum and Penicillium expansum isolated on the board of the Mir space station ]. / Biosintez nizkomolekuliarnykh azotsoderzhashchikh vtorichnykh metabolitov-alkaloidov shtammami-rezidentami gribov vidov Penicillium chrysogenum i Penicillium expansum, vydelennykh na orbital'noi stantsii "Mir".

The analysis of the absorption spectra of the low-molecular-weight nitrogen-containing secondary metabolites--alkaloids--of 4 Penicillium chrysogenum strains and 6 Penicillium expansum strains isolated on board the Mir space station showed that all these strains synthesize metabolites of alkaloid origin (roquefortine, 3,12-dihydroroquefortine, meleagrin, viridicatin, viridicatol, isorugulosuvin, rugulosuvin B, N-acetyl-tryptamine, and a "yellow metabolite" containing the benzoquinone chromophore).

Secretory production of chicken ovomucoid domain 3 by Escherichia coli and alteration of inhibitory specificity toward proteases by substitution of the P1 site residue.

Ovomucoids are commonly present in bird egg white and exhibit inhibitory activity toward various serine proteases. To investigate the structure-function relationship of ovomucoid domain 3, we established a secretory expression system for the chicken ovomucoid domain 3 (OMCHI3)-encoding gene in Escherichia coli by ligating it downstream from the tac promoter and signal peptide of E. coli alkaline phosphatase. E. coli JM105 was transformed with the resulting plasmid and induced with 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG). The mature OMCHI3 was detected in the culture supernatant, and was purified to homogeneity by three-step chromatography. Amino-acid sequence analysis showed that processing by the signal peptidase was carried out exactly at the expected site. Measurements of circular dichroism spectra and inhibitory activity indicated that OMCHI3 was produced in the properly folded form. Furthermore, site-specific replacement of the Ala residue at the P1 site with Met or Lys resulted in acquisition of inhibitory activity toward chymotrypsin or trypsin, respectively, indicating that the P1 site is the predominant determinant for inhibitory specificity.

Overexpression and purification of avian ovomucoid third domains in Escherichia coli.

Genetic engineering studies of ovomucoid domains have been hindered by the lack of an efficient procedure for overproducing this protein. The novel scheme presented here has led to the isolation of chicken ovomucoid third domain (OMCHI3) at a level of 22 mg pure protein/l Escherichia coli culture medium. The gene coding for OMCHI3 was fused to the 3' end of the gene encoding staphylococcal nuclease (SNase). Expression of the chimeric gene was placed under control of the strong transcription and translation signals of the phage T7 promoter. Upon isopropyl-beta-D-galactopyranoside induction, the cells harboring the target plasmid efficiently overproduced the protein (30% of the total soluble protein). The 56-residue fragment corresponding to OMCHI3 was then liberated by cyanogen bromide (CNBr) cleavage at a genetically engineered methionine residue located at the nuclease--OMCHI3 junction (OMCHI3 lacks an internal methionine). SDS--PAGE, enzyme inhibition studies and NMR spectroscopy all indicated that the recombinant OMCHI3 has properties identical to those of OMCHI3 isolated from its natural source. The expression system was easily adapted for the production of [98% U 15N] OMCHI3. The expression vector was mutated for overexpression of turkey ovomucoid third domain (OMTKY3), which differs from OMCHI3 by three amino acid substitutions. Since many other avian ovomucoid domains also lack methionine residues, this approach should be suitable for large-scale production and isotope labeling of homologous proteinase inhibitors with a variety of inhibitory specificities.

The structure of sugar chains of Japanese quail ovomucoid. The occurrence of oligosaccharides not expected from the classical biosynthetic pathway for N-glycans; a method for the assessment of the structure of glycans present in picomolar amounts.

While the structure of the major oligosaccharide of Japanese quail ovomucoid was reported earlier (Hase, S. et al. (1982) J. Biochem. 91, 735-737), the structures of the minor oligosaccharide units were investigated for the first time in the present studies. For this purpose, the glycans of the protein were liberated from the polypeptide chain by hydrazinolysis. After N-acetylation, the reducing ends of the oligosaccharides obtained were coupled with 2-aminopyridine, and then the resulting fluorescent derivatives were purified by Bio-Gel P-2 column chromatography and reversed-phase HPLC. The chemical structures of two minor oligosaccharide units were determined with the aid of exoglycosidases, and by methylation analysis and Smith degradation. The results demonstrated that the ovomucoid contains the following two monoantennary glycans: Man alpha 1-6(Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc and Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc. The latter structure was not predicted by the classical metabolic pathway for the N-glycans to be formed. The structures of three additional minor heterosaccharides were deduced from their elution positions on HPLC together with the results of determination of their molecular sizes and the HPLC elution positions of their enzymatic degradation products. It is noteworthy that for the latter procedure for the estimation of the structures of oligosaccharides only minute quantities of glycans (several hundreds pmol) are required.

Effects of serial hormone treatments on egg white protein synthesis. Further evidence of translational regulation.

The administration of either progesterone or estrogen to withdrawn chicks several hours after a first dose of estrogen affected ovalbumin synthesis differently than its mRNA levels [S. S. Seaver (1981) J. steroid Biochem. 14, 949-957]. This suggested that the hormones were regulating the translation of ovalbumin directly. In this paper we report that serial hormone treatments also affect the rates of synthesis of two other egg white proteins, conalbumin and ovomucoid. When progesterone was administered 4 h after estrogen, conalbumin synthesis decreased. When either progesterone or a second dose of estrogen was administered 12 h after the first dose of estrogen, conalbumin synthesis increased. Serial hormone treatments did not always affect all three proteins similarly. At later times, administering progesterone after estrogen decreased ovomucoid synthesis but did not affect conalbumin or ovalbumin synthesis. To determine if the serial hormone treatments affect egg white protein mRNA's in a similar way, changes in ovalbumin and conalbumin mRNA levels were quantified in a rabbit reticulocyte cell-free translation system and were compared to changes in ovalbumin and conalbumin synthesis as measured in chick oviduct tissue minces. When serial hormone treatments were 12 h apart, ovalbumin and conalbumin synthesis was 50-300% higher than that predicted by the changes in ovalbumin or conalbumin mRNA levels. This is further evidence that translation of both conalbumin mRNA and ovalbumin mRNA is directly regulated by steroid hormones.

The signal sequence of ovalbumin is located near the NH2 terminus.

Ovalbumin, unlike other secretory proteins, is synthesized and secreted without cleavage of a hydrophobic signal peptide. Kinetic experiments were performed in a cell-free translation system to measure the minimum size of ovalbumin nascent chains required for binding of both the nascent chain and the corresponding mRNA to microsomal membranes derived from dog pancreas. Results of these experiments revealed that 50 to 60 amino acid residues are sufficient to bind ovalbumin-synthesizing polysomes to membranes in vitro. When microsomes with associated polysomes were isolated from chick oviduct, nascent ovalbumin chains longer than 50 residues were protected from proteolysis as long as the membranes remained intact, suggesting that the polypeptides were sequestered by the endoplasmic reticulum. We conclude that the functional signal for membrane translocation of ovalbumin becomes accessible when the nascent chain is 50 to 60 residues long. We speculate that the hydrophobic sequence which lies between residues 25 and 45 folds back on the preceding residues to form an amphipathic hairpin structure which is the signal element recognized by the membrane.

Ribonucleic acid precursors are associated with the chick oviduct nuclear matrix.

Nuclear matrix was prepared by sequential treatment of oviduct nuclei with Triton X-100, DNase I, and 2 M NaCl. Published procedures were modified such that as many steps as possible were performed at -20 degrees C to minimize endogenous ribonuclease activity. Examination of electron micrographs confirmed the isolation of intact nuclear matrix structures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins in these structures showed an absence of histones and an enrichment of certain nonhistone proteins. RNA was isolated from the nuclear matrix preparations and subjected to denaturing gel electrophoresis. Gels were analyzed by ethidium bromide staining and by hybridization of Northern blots to cloned DNA probes for ovalbumin, ovomucoid, 5.8S ribosomal RNA, and U1 RNA. All of the precursors to ovalbumin and ovomucoid mRNAs (including various splicing intermediates) and all of the precursors to ribosomal RNA were associated exclusively with the nuclear matrix fraction. By contrast, mature ovalbumin and ovomucoid mRNAs were distributed between matrix and nonmatrix fractions. These observations were further supported by quantitative hybridization analysis of the RNA in nuclear and matrix fractions. It was found that less than 50% of the mature message of intact nuclei was recovered in the matrix, while most significantly, over 95% of the mRNA precursors remained associated with the matrix. Finally, mature ribosomal RNAs and virtually all of the small nuclear RNAs (including U1 RNA) were also distributed between matrix and nonmatrix fractions. Our results suggest that all precursor RNAs (be they precursors to mRNA or rRNA) are exclusively associated with the nuclear matrix and support the notion that the nuclear matrix may be the structural site for RNA processing within the nuclei of eucaryotic cells.

Processing of precursor proteins by preparations of oviduct microsomes.

The signal peptidases from both chicken oviduct and dog pancreas microsomes have been detected by a post-translational assay and, in the case of dog microsomes, by a cotranslational assay. This proteolytic activity is not sensitive to a variety of conventional protease inhibitors under the conditions used in these assays. Using a synthetic ester substrate, two additional activities have been observed in oviduct microsomes, but neither appears to correspond to the signal peptidase activity. Using cDNA as a probe for specific mRNAs, it was shown that polysomes, in the process of synthesizing secretory proteins, bind to microsomal membrane vesicles. This binding appears to be dependent on the nature of the translation product (presumably the specific signal peptide) rather than on the mRNA itself. In addition, pretreatment of the membrane vesicles with N-ethyl maleimide prevents the vectorial discharge and processing of several secretory proteins but does not inhibit the binding of their polysomes to membranes.

Isolation and characterization of the chicken ovomucoid gene.

The chicken ovomucoid gene has been isolated by screening a chicken DNA library with a plasmid containing ovomucoid mRNA sequences. Twelve recombinant phages carrying ovomucoid mRNA sequences were isolated. Two of them, extending farthest into the 5' and 3' direction respectively, were characterized by restriction mapping and Southern hybridization as well as by electron microscopic analysis of hybrids between the cloned DNA and ovomucoid mRNA. Seven intervening sequences interrupt the ovomucoid mRNA sequence in chromosomal DNA. From these data a minimal size of 5.6 kb can be estimated for the length of the ovomucoid gene.

Isolation of recombinant plasmids bearing cDNA to hen ovomucoid and lysozyme mRNAs.

A large library of hen oviduct cDNA-pCR1 recombinant plasmids has been established in Escherichia coli X1776. From this library, ovomucoid cDNA and lysozyme cDNA-bearing plasmids have been identified. One of these plasmids, pMu7, yielded the sequence of the 3'-untranslated region of ovomucoid mRNA.