Novel fatty acid methyl esters from the actinomycete Micromonospora aurantiaca

The volatiles released by Micromonospora aurantiaca were collected by means of a closed-loop stripping apparatus (CLSA) and analysed by GC–MS. The headspace extracts contained more than 90 compounds from different classes. Fatty acid methyl esters (FAMEs) comprised the major compound class including saturated unbranched, monomethyl and dimethyl branched FAMEs in diverse structural variants: Unbranched, α-branched, γ-branched, (ω−1)-branched, (ω−2)-branched, α- and (ω−1)-branched, γ- and (ω−1)-branched, γ- and (ω−2)-branched, and γ- and (ω−3)-branched FAMEs. FAMEs of the last three types have not been described from natural sources before. The structures for all FAMEs have been suggested based on their mass spectra and on a retention index increment system and verified by the synthesis of key reference compounds. In addition, the structures of two FAMEs, methyl 4,8-dimethyldodecanoate and the ethyl-branched compound methyl 8-ethyl-4-methyldodecanoate were deduced from their mass spectra. Feeding experiments with isotopically labelled [2H10]leucine, [2H10]isoleucine, [2H8]valine, [2H5]sodium propionate, and [methyl-2H3]methionine demonstrated that the responsible fatty acid synthase (FAS) can use different branched and unbranched starter units and is able to incorporate methylmalonyl-CoA elongation units for internal methyl branches in various chain positions, while the methyl ester function is derived from S-adenosyl methionine (SAM)

Conformational Analysis, Thermal Rearrangement, and EI-MS Fragmentation Mechanism of (1(10)E,4E,6S,7R)-Germacradien-6-ol by ¹³C-Labeling Experiments

An uncharacterized terpene cyclase from Streptomyces pratensis was identified as (+)-(1(10)E,4E,6S,7R)-germacradien-6-ol synthase. The enzyme product exists as two interconvertible conformers, resulting in complex NMR spectra. For the complete assignment of NMR data, all fifteen (13C1)FPP isotopomers (FPP=farnesyl diphosphate) and (13C15)FPP were synthesized and enzymatically converted. The products were analyzed using various NMR techniques, including 13C, 13C COSY experiments. The (13C)FPP isotopomers were also used to investigate the thermal rearrangement and EI fragmentation of the enzyme product.publishe

Biochemistry and Crystal Structure of Ectoine Synthase: A Metal-Containing Member of the Cupin Superfamily.

Ectoine is a compatible solute and chemical chaperone widely used by members of the Bacteria and a few Archaea to fend-off the detrimental effects of high external osmolarity on cellular physiology and growth. Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction. However, the crystal structure of ectoine synthase is not known and a clear understanding of how its fold contributes to enzyme activity is thus lacking. Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form. Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily. EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure. The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer. We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme. We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency. Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants. An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel

Conversion of N-γ-ADABA into ectoine by (<i>Sa</i>)EctC mutant derivatives and their iron-content.

<p>Conversion of N-γ-ADABA into ectoine by (<i>Sa</i>)EctC mutant derivatives and their iron-content.</p

Dependency of the ectoine synthase activity on metals.

<p>(a) Impact of the iron-chelator EDTA on the enzyme activity of the purified (<i>Sa</i>)EctC protein. Metal depletion and reconstitution experiments with (b) stoichiometric and (c) excess amounts of metals. The (<i>Sa</i>)EctC protein was present at a concentration of 10 μM. The level of enzyme activity given in (b) is benchmarked relative to that of ectoine synthase enzyme assays in which 1 mM FeCl₂ was added.</p

A Single Sfp-Type Phosphopantetheinyl Transferase Plays a Major Role in the Biosynthesis of PKS and NRPS Derived Metabolites in <i>Streptomyces ambofaciens</i> ATCC23877

<div><p>The phosphopantetheinyl transferases (PPTases) are responsible for the activation of the carrier protein domains of the polyketide synthases (PKS), non ribosomal peptide synthases (NRPS) and fatty acid synthases (FAS). The analysis of the <i>Streptomyces ambofaciens</i> ATCC23877 genome has revealed the presence of four putative PPTase encoding genes. One of these genes appears to be essential and is likely involved in fatty acid biosynthesis. Two other PPTase genes, <i>samT0172</i> (<i>alpN</i>) and <i>samL0372</i>, are located within a type II PKS gene cluster responsible for the kinamycin production and an hybrid NRPS-PKS cluster involved in antimycin production, respectively, and their products were shown to be specifically involved in the biosynthesis of these secondary metabolites. Surprisingly, the fourth PPTase gene, which is not located within a secondary metabolite gene cluster, appears to play a pleiotropic role. Its product is likely involved in the activation of the acyl- and peptidyl-carrier protein domains within all the other PKS and NRPS complexes encoded by <i>S. ambofaciens</i>. Indeed, the deletion of this gene affects the production of the spiramycin and stambomycin macrolide antibiotics and of the grey spore pigment, all three being PKS-derived metabolites, as well as the production of the nonribosomally produced compounds, the hydroxamate siderophore coelichelin and the pyrrolamide antibiotic congocidine. In addition, this PPTase seems to act in concert with the product of <i>samL0372</i> to activate the ACP and/or PCP domains of the antimycin biosynthesis cluster which is also responsible for the production of volatile lactones.</p></div

Crystal structure of (<i>Sa</i>)EctC.

<p>(a) Top-view of the dimer of the (<i>Sa</i>)EctC protein. The position of the water molecule, described in detail in the text, is shown in one of the monomers as an orange sphere. (b) Side-view of a (<i>Sa</i>)EctC dimer allowing an assessment of the dimer interface formed by two β-strands of each monomer. (c) Close-up representation of the dimer interface mediated by beta-strand β1 and β6.</p

A chemically undefined ligand is captured in the active site of the “semi-closed” (<i>Sa</i>)EctC crystal structure.

<p>(a) The observed electron density in the active site of the “semi-closed” structure of (<i>Sa</i>)EctC is modeled as a hexane-1,6-diol molecule and compared with the electron density of the <i>N</i>-γ-ADABA substrate of the ectoine synthase to emphasize the similarity in size of these compounds. (b) The presumable binding site of the iron co-factor and of the modeled hexane-1,6-diol molecule is depicted. The amino acid side chains involved in iron-ligand binding are colored in blue and those involved in the binding of the chemically undefined ligand are colored in green using a ball and stick representation. The flexible carboxy-terminal loop of (<i>Sa</i>)EctC is highlighted in orange. The electron density was calculated as an omit map and contoured at 1.0 σ.</p

Overall structure of the “open” and “semi-closed” crystal structures of (<i>Sa</i>)EctC.

<p>(a) The overall structure of the “semi-closed” (<i>Sa</i>)EctC resolved at 2.0 Å is depicted in green in a cartoon (upper panel) and surface (lower panel) representation. The β-strands are numbered β1-β11 and the helices α-I to α-II. (b) The overall structure of the “open” (<i>Sa</i>)EctC was resolved at 1.2 Å and is depicted in yellow in a cartoon (upper panel) and surface (lower panel) representation. The entrance to the active site of the ectoine synthase is marked. (c) Overlay of the “semi-closed” and “open” (<i>Sa</i>)EctC structures.</p