Crystal structure of Acetyl-CoA carboxylase (AccB) from Streptomyces antibioticus and insights into the substrate-binding through in silico mutagenesis and biophysical investigations.

Acetyl-CoA carboxylase (ACC) is crucial for polyketides biosynthesis and acts as an essential metabolic checkpoint. It is also an attractive drug target against obesity, cancer, microbial infections, and diabetes. However, the lack of knowledge, particularly sequence-structure function relationship to narrate ligand-enzyme binding, has hindered the progress of ACC-specific therapeutics and unnatural "natural" polyketides. Structural characterization of such enzymes will boost the opportunity to understand the substrate binding, designing new inhibitors and information regarding the molecular rules which control the substrate specificity of ACCs. To understand the substrate specificity, we determined the crystal structure of AccB (Carboxyl-transferase, CT) from Streptomyces antibioticus with a resolution of 2.3 Å and molecular modeling approaches were employed to unveil the molecular mechanism of acetyl-CoA recognition and processing. The CT domain of S. antibioticus shares a similar structural organization with the previous structures and the two steps reaction was confirmed by enzymatic assay. Furthermore, to reveal the key hotspots required for the substrate recognition and processing, in silico mutagenesis validated only three key residues (V223, Q346, and Q514) that help in the fixation of the substrate. Moreover, we also presented atomic level knowledge on the mechanism of the substrate binding, which unveiled the terminal loop (500-514) function as an opening and closing switch and pushes the substrate inside the cavity for stable binding. A significant decline in the hydrogen bonding half-life was observed upon the alanine substitution. Consequently, the presented structural data highlighted the potential key interacting residues for substrate recognition and will also help to re-design ACCs active site for proficient substrate specificity to produce diverse polyketides.

Tat-Dependent Heterologous Secretion of Recombinant Tyrosinase by Pseudomonas fluorescens Is Aided by a Translationally Fused Caddie Protein.

Tyrosinase is a monooxygenase that catalyzes both the hydroxylation of p-hydroxyphenyl moieties to o-catechols and the oxidation of o-catechols to o-quinones. Apart from its critical functionality in melanogenesis and the synthesis of various neurotransmitters, this enzyme is also used in a variety of biotechnological applications, most notably mediating covalent cross-linking between polymers containing p-hydroxyphenyl groups, forming a hydrogel. Tyrosinases from the genus Streptomyces are usually secreted as a complex with their caddie protein. In this study, we report an increased secretion efficiency observed when the Streptomyces antibioticus tyrosinase gene melC2 was introduced into Pseudomonas fluorescens along with its caddie protein gene melC1, which has the DNA sequence for the Tat (twin-arginine translocation) signal.IMPORTANCE We observed that the S. antibioticus extracellular tyrosinase secretion level was even higher in its nonnatural translationally conjugated fusion protein form than in the natural complex of two separated polypeptides. The results of this study demonstrate that tyrosinase-expressing P. fluorescens can be a stable source of bacterial tyrosinase through exploiting the secretory machinery of P. fluorescens.

Insights into a divergent phenazine biosynthetic pathway governed by a plasmid-born esmeraldin gene cluster.

Phenazine-type metabolites arise from either phenazine-1-carboxylic acid (PCA) or phenazine-1,6-dicarboxylic acid (PDC). Although the biosynthesis of PCA has been studied extensively, PDC assembly remains unclear. Esmeraldins and saphenamycin, the PDC originated products, are antimicrobial and antitumor metabolites isolated from Streptomyces antibioticus Tü 2706. Herein, the esmeraldin biosynthetic gene cluster was identified on a dispensable giant plasmid. Twenty-four putative esm genes were characterized by bioinformatics, mutagenesis, genetic complementation, and functional protein expressions. Unlike enzymes involved in PCA biosynthesis, EsmA1 and EsmA2 together decisively promoted the PDC yield. The resulting PDC underwent a series of conversions to give 6-acetylphenazine-1-carboxylic acid, saphenic acid, and saphenamycin through a unique one-carbon extension by EsmB1-B5, a keto reduction by EsmC, and an esterification by EsmD1-D3, the atypical polyketide sythases, respectively. Two transcriptional regulators, EsmT1 and EsmT2, are required for esmeraldin production.

Inhibition of oxidative stress-induced amyloid beta formation in NT2 neurons by culture filtrate of a strain of Streptomyces antibioticus.

Actinomycetes isolated from Iran soil habitats were tested for the capacity to produce compounds which can protect neurons from cell death generated by oxidative stress in NT2 neurons. Confirmation of our initial hit was accomplished via the determination of amyloid beta level using the enzyme-linked immunosorbent assay test. The most interesting amyloid beta formation inhibitor discovered in our study was a secondary metabolite which was produced by strain HM45. This bioactive strain was identified as a strain of Streptomyces antibioticus DSM 40234 using polyphasic approach. The strain HM45 was deposited in Deutsche Sammlung von Mikroorganismen und Zellkulturen as S. antibioticus DSM 41955 and University of Tehran Microorganisms Sollection as S. antibioticus UTMC 00105. This work is the first report on efficiency of an actinomycete metabolite in prohibition of neurons death caused by amyloid beta formation.

Genetic organization of the biosynthetic gene cluster for the antitumor angucycline oviedomycin in Streptomyces antibioticus ATCC 11891.

The oviedomycin biosynthetic gene cluster from Streptomyces antibioticus ATCC 11891 has been sequenced and characterized. It contains all the necessary genes for oviedomycin biosynthesis, together with several genes for the generation of malonyl-CoA extender units. Production of this unusual angucyclinone in its natural host occurs only in solid cultures in parallel with aerial mycelium and spore formation. A mutant that did not produce oviedomycin was generated by disruption of the beta-ketoacyl synthase gene ovmK. No other physiological process in the mutant appears to be affected; this rules out a direct relationship between oviedomycin production and cell differentiation in S. antibioticus.

Simocyclinones: diversity of metabolites is dependent on fermentation conditions.

Simocyclinones, a novel group of angucyclinone antibiotics, are produced by Streptomyces antibioticus Tü 6040. The compounds show antibacterial and antitumor properties. In submerged cultivation, the production of simocyclinones is strongly dependent on the carbon and nitrogen sources used in a chemically defined medium. Productivity of distinct components and diversity of simocyclinone compounds are influenced by the medium composition. Four series of simocyclinone compounds were detected by high-performance liquid chromatography (HPLC) diode array detector (DAD) and HPLC electrospray ionization (ESI) mass spectrometry (MS) analysis, isolated and the structures determined by nuclear magnetic resonance (NMR) techniques. Under optimized conditions, simocyclinone D8 was produced in an amount of 300 mg l(-1) and simocyclinone C4 in a concentration up to 50 mg l(-1).

Simocyclinones, novel cytostatic angucyclinone antibiotics produced by Streptomyces antibioticus Tü 6040. I. Taxonomy, fermentation, isolation and biological activities.

Two novel angucyclinone-type antibiotics, simocyclinones D4 and D8, were detected in the mycelium extract of Streptomyces antibioticus Tü 6040 by HPLC-diode-array and HPLC-electrospray-mass-spectrometry screening. The compounds show antibiotic activities against Gram-positive bacteria and cytostatic effects on various tumor cell lines.

Microbial growth and production kinetics of Streptomyces antibioticus Tü 6040.

Streptomyces antibioticus Tü 6040 is the producer of simocyclinones, which belong to a novel family of angucyclinone antibiotics some of which show antitumor activities. Growth and antibiotic production is dependent on the medium composition, especially on the carbon and nitrogen source, and on the fermentation conditions. The best results with respect to antibiotic productivity were achieved using a chemically defined medium with glycerol and L-lysine as carbon and nitrogen source, respectively, in an airlift fermenter with minimised shear stress at low gas flow rates withour oxygen limitation. These conditions led to a homogeneous formation of pellets of 1-2 mm in diameter and guaranteed reproducible product yields of the main compound, simocyclinone D8, in the range of 300 mg/l.

relA is required for actinomycin production in Streptomyces antibioticus.

The relA gene from Streptomyces antibioticus has been cloned and sequenced. The gene encodes a protein with an Mr of 93,653, which is 91% identical to the corresponding protein from Streptomyces coelicolor. Disruption of S. antibioticus relA produces a strain which grows significantly more slowly on actinomycin production medium than the wild type or a disruptant to which the intact relA gene was restored. Moreover, the disruptant was unable to accumulate ppGpp to the levels observed during the normal course of growth and actinomycin production in the wild type. The strain containing the disrupted relA gene did not produce actinomycin and contained significantly lower levels of the enzyme phenoxazinone synthase than the wild-type strain. Actinomycin synthetase I, a key enzyme in the actinomycin biosynthetic pathway, was undetectable in the relA disruptant. Growth of the disruptant on low-phosphate medium did not restore actinomycin production.

Measurement of the degree of coupled isotopic enrichment of different positions in an antibiotic peptide by NMR.

An experimental strategy for determining the extent to which multiply isotopically labeled fragments are incorporated intact into relatively complicated compounds of interest is presented. The NMR methods employed are based on isotope-filtered one-dimensional spectra and difference HSQC spectra incorporating a spin echo designed to report on the presence of a second NMR active isotope at a coupled site. They supplement existing methods for determining the extent of isotopic incorporation at individual sites to reveal whether two coupled labeled sites in a precursor are incorporated as an intact unit into products. The methods described also circumvent 1H signal overlap and distinguish between the effects of different nitrogens coupled to individual carbons. The somewhat complicated case of valclavam illustrates the method's utility in measuring the J coupling constants between 13C and nearby sites that are only fractionally labeled with 15N, and measuring the fraction of molecules in which 13C is coupled to 15N, at each of several sites. The 15N of [2-13C, 15N]-labeled glycine is found to be incorporated into all three N positions of valclavam but most heavily into the N11 position. Specifically, 15N and 13C are incorporated into the N11 and C10 positions together as an 15N13C fragment approximately 8% of the time, whereas 15N is incorporated largely independently at the other positions.

Characterization of the ATPase activity of the N-terminal nucleotide binding domain of an ABC transporter involved in oleandomycin secretion by Streptomyces antibioticus.

The oleB gene of Streptomyces antibioticus, oleandomycin producer, encodes an ABC transporter containing two putative ATP-binding domains and is involved in oleandomycin resistance and secretion in this organism. We have overexpressed in Escherichia coli the N-terminal nucleotide-binding domain of OleB (OleB') as a fusion protein and purified the fusion protein by affinity chromatography. The fusion protein showed ATPase activity dependent on the presence of Mg2+ ions. ATPase activity was resistant to specific inhibitors of P-, F-, and V-type ATPase whereas sodium azide and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) were strong inhibitors. The change of Lys71, located within the Walker A motif of the OleB' protein, to Gln or Glu caused a loss of ATPase activity, whereas changing to Gly did not impair the activity. The results suggest that the intrinsic ATPase activity of purified fusion protein can be clearly distinguished from other ATP-hydrolysing enzymes, including ion-translocating ATPases or ABC-traffic ATPases, both on the basis of inhibition by different agents and since it hydrolyzes ATP without interacting with a hydrophobic membrane component.

Effects of 5-azacytidine on physiological differentiation of Streptomyces antibioticus.

We studied the specificity of the effect of 5-azacytidine, a DNA-methylase inhibitor that impairs Streptomyces differentiation. We showed that this compound did not affect global DNA, RNA or protein biosynthesis in submerged cultures of S. antibioticus ETHZ 7451. Among individual proteins, enzymes such as alkaline phosphatase and intracellular protease were produced in similar amounts in the presence and absence of this compound. However, the production of extracellular protease was significantly inhibited. Also DNA-methyltransferases were inhibited, indicating that DNA methylation might be involved in the regulation of differentiation. By contrast, elevated levels of the antibiotic rhodomycin resulted when 5-azacytidine was added to the culture medium. In order to determine whether there was a correlation between sporulation and altered enzymatic activities, these activities were analysed in S. antibioticus submerged cultures. Among them, alkaline phosphatase and intracellular protease activities did not show a clear correlation with sporulation. However, high levels of extracellular protease were produced during septation of hyphae. This association between extracellular protease and sporulation suggests a specific inhibitory effect of 5-azacytidine, not only on spore formation, but also on physiological differentiation.

Streptomyces antibioticus contains at least three oleandomycin-resistance determinants, one of which shows similarity with proteins of the ABC-transporter superfamily.

Three different DNA fragments of an oleandomycin producer, Streptomyces antibioticus, conferring oleandomycin resistance were cloned in plasmid pIJ702 and expressed in Streptomyces lividans and in Streptomyces albus. These oleandomycin resistance determinants were designated as oleA (pOR400), oleB (pOR501) and oleC (pOR800). oleA and oleC are closely linked in the chromosome as they were both obtained together in two cosmid clones that were isolated from a genomic library. Sequencing of the oleC resistance determinant revealed four complete open reading frames (ORFs) and the C-terminal end of a fifth. The functions of orf1 and orf2 are unknown since they did not show significant similarity with other sequences in the data bases. The orf3 gene product has similarity with some proteins involved in iron and vitamin B12 uptake in bacteria. The orf4 gene product had a hydrophilic profile and showed important similarity with proteins containing typical ATP-binding domains characteristic of the ABC-transporter superfamily and involved in membrane transport and, particularly, with several genes conferring resistance to various macrolide antibiotics and anticancer drugs. The last gene, orf5, is translationally coupled to orf4 and codes for a hydrophobic polypeptide containing several transmembrane domains characteristic of integral membrane proteins. Subcloning and deletion experiments limited the resistance determinant to a 0.9 kb PstI-SphI fragment and only orf4 is included in this fragment. These results suggest that resistance to oleandomycin conferred by oleC (orf4) is probably due to an efflux transport system of the ABC-transporter superfamily.

[Effect of the producer type on the nature of antibiotic fermentation]. / Vliianie tipa produtsenta na kharakter fermentatsii antibiotikov.

Fermentation processes in production of bacitracin, a polypeptide antibiotic by Bacillus licheniformis, and oleandomycin, a macrolide antibiotic by Streptomyces antibioticus, were studied comparatively. It was shown that the antibiotic-producing actinomycete was characterized by a prolonged phase of growth retardation. The highest efficiency of the control actions was observed at the beginning of the fermentation. They were aimed at intensifying the substrate usage during the growth phase and activation of cell metabolism. Controlled cultivation of the Bacillus representative was based on its capacity of achieving the maximum growth rate possible under the certain conditions. Therefore, an increase in the quantity of the synthesized antibiotic was due, under such conditions, to inhibition of the culture growth by various means including lower mass exchange intensity.

Biosynthesis of 9-beta-D-arabinofuranosyladenine: hydrogen exchange at C-2' and oxygen exchange at C-3' of adenosine.

The data presented here describe new findings related to the bioconversion of adenosine to 9-beta-D-arabinofuranosyladenine (ara-A) by Streptomyces antibioticus by in vivo investigations and with a partially purified enzyme. First, in double label in vivo experiments with [2'-18O]- and [U-14C]adenosine, the 18O:14C ratio of the ara-A isolated does not change appreciably, indicating a stereospecific inversion of the C-2' hydroxyl of adenosine to ara-A with retention of the 18O at C-2'. In experiments with [3'-18O]- and [U-14C]-adenosine, [U-14C]ara-A was isolated; however, the 18O at C-3' is below detection. The adenosine isolated from the RNA from both double label experiments has essentially the same ratio of 18O:14C. Second, an enzyme has been isolated and partially purified from extracts of S. antibioticus that catalyzes the conversion of adenosine, but not AMP, ADP, ATP, inosine, guanosine, or D-ribose, to ara-A. In a single label enzyme-catalyzed experiment with [U-14C]adenosine, there was a 9.9% conversion to [U-14C]ara-A; with [2'-3H]-adenosine, there was a 8.9% release of the C-2' tritium from [2'-3H]adenosine which was recovered as 3H2O. Third, the release of 3H as 3H2O from [2'-3H]adenosine was confirmed by incubations of the enzyme with 3H2O and adenosine. Ninety percent of the tritium incorporated into the D-arabinose of the isolated ara-A was in C-2 and 8% was in C-3. The enzyme-catalyzed conversion of adenosine to ara-A occurs without added cofactors, displays saturation kinetics, a pH optimum of 6.8, a Km of 8 X 10(-4) M, and an inhibition by heavy metal cations. The enzyme also catalyzes the stereospecific inversion of the C-2' hydroxyl of the nucleoside antibiotic, tubercidin to form 7-beta-D-arabinofuranosyl-4-aminopyrrolo[2,3-d]pyrimidine. The nucleoside antibiotic, sangivamycin, in which the C-5 hydrogen is replaced with a carboxamide group, is not a substrate. On the basis of the single and double label experiments in vivo and the in vitro enzyme-catalyzed experiments, two mechanisms involving either a 3'-ketonucleoside intermediate or a radical cation are proposed to explain the observed data.

Incremento en la producción de eritromicina por fusión de protoplastos de Streptomyces erythreus / Increasing in the production of erythromycin by fusion of protoplasts of Streptomyces erythreus

El método de fusión de protoplastos fue usado para la obtención de recombinantes con producción antibiótica incrementada en Streptomyces erythreus productora de eritromicina. Se emplearon los mutantes morfológicos 36 UV y P-E obtenidos por luz UV y luz UV-acriflavina respectivamente, los cuales poseían marcadores de resistencia a cloranfenicol y rifampicina. Se establecen las condiciones más apropiadas para la obtención y fusión de protoplastos en estas cepas, llegándose a obtener recombinantes resistentes a ambos antibióticos a frecuencia de 0,57 %, dentro de las cuales fue seleccionado uno con incremento de 4 veces en la producción de eritromicina

8-Ketodeoxycoformycin and 8-ketocoformycin as intermediates in the biosynthesis of 2'-deoxycoformycin and coformycin.

An enzyme has been isolated from cell-free extracts of Streptomyces antibioticus that can catalyze the reduction of 8-ketodeoxycoformycin (8-KetodCF) and 8-ketocoformycin (8-ketoCoF) to the naturally occurring nucleoside analogues 2'-deoxycoformycin (dCF) and coformycin (CoF), respectively. The partially purified reductase requires NADPH as the cofactor and stereospecifically reduces the 8-keto group of both ketonucleoside substrates to a hydroxyl group with the R configuration at C-8. This is the same configuration of the hydroxyl group as that of the dCF and CoF isolated from S. antibioticus. The reduction proceeds at the nucleoside level, and ATP is not required. The reductase is stereospecific for the NADPH cofactor in that it transfers the pro-S but not the pro-R hydrogen from C-4 of NADPH to the 8-keto group. The apparent Km for 8-ketodCF and 8-ketoCoF were 250 and 150 microM, respectively. These in vitro results, which show that 8-ketodCF and 8-ketoCoF may be intermediates in the biosynthesis of dCF and CoF, support and extend our earlier results from in vivo studies which established that adenosine and C-1 of D-ribose are the carbon-nitrogen precursors of dCF. A possible mechanism for the formation of dCF is presented.

[Effect of surface-active agents (tween-21) on indices of energy metabolism in oleandomycin producers]. / Vliianie poverkhnostno-aktivnykh veshchestv (tvina-21) na pokazateli énergeticheskogo obmena u produtsenta oleandomitsina.

The specific growth rate of Streptomyces antibioticus, a producer of oleandomycin, and the specific rate of the antibiotic accumulation in the culture medium during fermentation were investigated. On the basis of the results obtained the fermentation period was divided into 7 phases of development. The culture treated with the surfactant (Tween-21) is characterized by a higher specific growth rate during the whole fermentation and a higher specific rate of the antibiotic accumulation at the stage of the highest production as compared to the control. The ATP content, the value of the adenylate energy charge and the contents of high-molecular weight polyphosphates in the mycelium were examined. In the phase of the intensive growth St. antibioticus was characterized by a higher ATP level and a higher energy charge. More active accumulation of polyphosphates was observed in the late intensive growth phase. It was also found that after the treatment of the culture with Tween-21 it utilized polyphosphates more actively during the antibiotic biosynthesis.

Actinomycin synthesis in Streptomyces antibioticus: enzymatic conversion of 3-hydroxyanthranilic acid to 4-methyl-3-hydroxyanthranilic acid.

A methyltransferase which utilizes 3-hydroxyanthranilic acid (HAA) as a substrate was identified in detergent-treated extracts of the bacterium Streptomyces antibioticus. The enzyme catalyzes the transfer of methyl groups from [14C]S-adenosylmethionine to HAA, but does not catalyze the methylation of 3-hydroxy-DL-kynurenine. Enzyme, substrate, time, and pH dependencies for the methyl transfer reaction were examined. Reaction products obtained from scaled-up reaction mixtures were fractionated by chromatography on Dowex 1, and the Dowex 1 fractions were examined by paper and thin-layer chromatography. One Dowex fraction was shown to contain a radioactive product with the chromatographic properties of 4-methyl-3-hydroxyanthranilic acid (MHA), a known intermediate in the biosynthesis of actinomycin. Available evidence indicates that the conversion of HAA to MHA is an early step in the biosynthesis of actinomycin by S. antibioticus and other actinomycin-producing streptomycetes.

[Inhibition of oleandomycin synthesis by glucoses added during fermentation]. / Podavlenie biosinteza oleandomitsina gliukozoi pri ee dobavlenii v protsesse fermentatsii.

Oleandomycin biosynthesis by Streptomyces antibioticus is repressed by glucose added to the growth medium in the process of fermentation. Phosphotransferase involved in the synthesis of acetyl CoA and propionyl CoA (the precursors of the antibiotic macrolactone ring) is neither inhibited nor repressed, and the substrate specificity of the enzyme does not change. The content of cAMP in the mycelium of S. antibioticus does not change significantly when either glucose or sucrose is added to the medium 24 h after the inoculation whereas the content of exogenous cAMP rises abruptly 24 h after glucose addition. At the same time, the medium becomes much more acidic and the content of protein in the mycelium rises noticeably. Consequently, cAMP may be involved in the regulation of the culture growth.