13 research outputs found
A Multienzyme Complex Channels Substrates and Electrons through Acetyl-CoA and Methane Biosynthesis Pathways in <i>Methanosarcina</i>
<div><p>Multienzyme complexes catalyze important metabolic reactions in many organisms, but little is known about the complexes involved in biological methane production (methanogenesis). A crosslinking-mass spectrometry (XL-MS) strategy was employed to identify proteins associated with coenzyme M-coenzyme B heterodisulfide reductase (Hdr), an essential enzyme in all methane-producing archaea (methanogens). In <i>Methanosarcina acetivorans</i>, Hdr forms a multienzyme complex with acetyl-CoA decarbonylase synthase (ACDS), and F<sub>420</sub>-dependent methylene-H<sub>4</sub>MPT reductase (Mer). ACDS is essential for production of acetyl-CoA during growth on methanol, or for methanogenesis from acetate, whereas Mer is essential for methanogenesis from all substrates. Existence of a Hdr:ACDS:Mer complex is consistent with growth phenotypes of ACDS and Mer mutant strains in which the complex samples the redox status of electron carriers and directs carbon flux to acetyl-CoA or methanogenesis. We propose the Hdr:ACDS:Mer complex comprises a special class of multienzyme redox complex which functions as a “biological router” that physically links methanogenesis and acetyl-CoA biosynthesis pathways.</p></div
Cyanohydrin Phosphonate Natural Product from <i>Streptomyces regensis</i>
<i>Streptomyces regensis</i> strain WC-3744 was identified
as a potential phosphonic acid producer in a large-scale screen of
microorganisms for the presence of the <i>pepM</i> gene,
which encodes the key phosphonate biosynthetic enzyme phosphoenolpyruvate
phosphonomutase. <sup>31</sup>P NMR revealed the presence of several
unidentified phosphonates in spent medium after growth of <i>S. regensis</i>. These compounds were purified and structurally
characterized via extensive 1D and 2D NMR spectroscopic and mass spectrometric
analyses. Three new phosphonic acid metabolites, whose structures
were confirmed by comparison to chemically synthesized standards,
were observed: (2-acetamidoethyl)Âphosphonic acid (<b>1</b>),
(2-acetamido-1-hydroxyethyl)Âphosphonic (<b>3</b>), and a novel
cyanohydrin-containing phosphonate, (cyanoÂ(hydroxy)Âmethyl)Âphosphonic
acid (<b>4</b>). The gene cluster responsible for synthesis
of these molecules was also identified from the draft genome sequence
of <i>S. regensis</i>, laying the groundwork for future
investigations into the metabolic pathway leading to this unusual
natural product
HdrD1 protein:protein interactions detected by Mass Spectrometry<sup>a</sup>.
a<p>Proteins also detected in the control samples have been omitted.</p>b<p>Proteins were identified in duplicate biological samples.</p><p>HdrD1 protein:protein interactions detected by Mass Spectrometry<sup><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107563#nt101" target="_blank">a</a></sup>.</p
Organization of cellular metabolism.
<p>Metabolic reactions in a cell can be catalyzed by <i>A</i>, individual enzymes, or <i>B</i>, multienzyme complexes that channel substrates and/or sequester intermediates in a pathway. Pathways in the cell can be connected in series, <i>C</i>, or in parallel by <i>D</i>, metabolic “routers” that channel electrons and substrates to either of two metabolic pathways.</p
XL-MS identification of a multienzyme complex in <i>Methanosarcina</i>.
<p><i>A</i>, Detection of HdrD complexes. 2 µg crosslinked cell lysates from controls or strains expressing strep-tagged HdrD1 protein were analyzed by Western blot. Arrows indicate the position of strep-tagged HdrD1 monomer and crosslinked high molecular weight (HMW) complexes. * = degraded HdrD1strep protein. <i>B</i>, HdrD co-purified proteins detected by mass spectrometry. Node sizes, line opacity and line widths are proportional to the average peptide hit score of the protein detected in biological duplicates. Dotted lines denote an average score below 100, solid lines denote an average score of 100 and above. Image created with Cytoscape <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107563#pone.0107563-Shannon1" target="_blank">[36]</a>. <i>C</i>, Putative model of the Hdr:ACDS:Mer complex. During methylotrophic growth, both ACDS and Mer use methyl-H<sub>4</sub>MPT as a substrate. Black dotted lines = electron flow between active sites. HdrE (blue) or HdrD (red), Mer is a tetramer (orange), and ACDS is composed of 5 subunits in a (α<sub>2</sub>ε<sub>2</sub>)<sub>4</sub>β<sub>8</sub>(γδ)<sub>8</sub> configuration (green) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107563#pone.0107563-Gong1" target="_blank">[13]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107563#pone.0107563-Aufhammer1" target="_blank">[16]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107563#pone.0107563-Kung1" target="_blank">[37]</a>.</p
Enzymes used by <i>M. acetivorans</i>.
<p>Current pathway models for growth of <i>M. acetivorans</i> on <i>A</i>, methanol + acetate or <i>B</i>, acetate as carbon and energy sources. Enzymes in red are essential despite no defined purpose in the pathway. Green ovals = energy conserving step. Red ovals = energy-consuming step. Please see text for abbreviations.</p
Comparison of methanogenesis pathways.
<p><i>A</i>, Hydrogenotrophic methanogenesis in <i>Methanococcus maripaludis. B</i>, Methylotrophic methanogenesis in <i>Methanosarcina acetivorans</i>. Green ovals: energy-conserving reactions. Red ovals: energy-consuming reaction. Please see text for abbreviations.</p
Cyanohydrin Phosphonate Natural Product from <i>Streptomyces regensis</i>
<i>Streptomyces regensis</i> strain WC-3744 was identified
as a potential phosphonic acid producer in a large-scale screen of
microorganisms for the presence of the <i>pepM</i> gene,
which encodes the key phosphonate biosynthetic enzyme phosphoenolpyruvate
phosphonomutase. <sup>31</sup>P NMR revealed the presence of several
unidentified phosphonates in spent medium after growth of <i>S. regensis</i>. These compounds were purified and structurally
characterized via extensive 1D and 2D NMR spectroscopic and mass spectrometric
analyses. Three new phosphonic acid metabolites, whose structures
were confirmed by comparison to chemically synthesized standards,
were observed: (2-acetamidoethyl)Âphosphonic acid (<b>1</b>),
(2-acetamido-1-hydroxyethyl)Âphosphonic (<b>3</b>), and a novel
cyanohydrin-containing phosphonate, (cyanoÂ(hydroxy)Âmethyl)Âphosphonic
acid (<b>4</b>). The gene cluster responsible for synthesis
of these molecules was also identified from the draft genome sequence
of <i>S. regensis</i>, laying the groundwork for future
investigations into the metabolic pathway leading to this unusual
natural product
Elucidating the Rimosamide-Detoxin Natural Product Families and Their Biosynthesis Using Metabolite/Gene Cluster Correlations
As
microbial genome sequencing becomes more widespread, the capacity
of microorganisms to produce an immense number of metabolites has
come into better view. Utilizing a metabolite/gene cluster correlation
platform, the biosynthetic origins of a new family of natural products,
the rimosamides, were discovered. The rimosamides were identified
in <i>Streptomyces rimosus</i> and associated with their
NRPS/PKS-type gene cluster based upon their high frequency of co-occurrence
across 179 strains of actinobacteria. This also led to the discovery
of the related detoxin gene cluster. The core of each of these families
of natural products contains a depsipeptide bond at the point of bifurcation
in their unusual branched structures, the origins of which are definitively
assigned to nonlinear biosynthetic pathways <i>via</i> heterologous
expression in <i>Streptomyces lividans</i>. The rimosamides
were found to antagonize the antibiotic activity of blasticidin S
against <i>Bacillus cereus</i>