17 research outputs found
Supporting information to the Figure 4
Phylogenetic relationships of 47 collection strains harboring erm resistance genes. An alignment of 16S rRNA gene sequences. A phylogram constructed by neighbor-joining method based on Jukes-Cantor distance matrices calculated from 1000 bootstrap replicates of the alignment
Diversity of Alkylproline Moieties in Pyrrolobenzodiazepines Arises from Postcondensation Modifications of a Unified Building Block
Anticancer pyrrolobenzodiazepines
(PBDs) are one of several groups
of natural products that contain unusual 4-alkyl-l-proline
derivatives (APDs) in their structure. APD moieties of PBDs are characterized
by high structural diversity achieved through unknown biosynthetic
machinery. Based on LC-MS analysis of culture broths, feeding experiments,
and protein assays, we show that APDs are not incorporated into PBDs
in their final form as was previously hypothesized. Instead, a uniform
building block, 4-propylidene-l-proline or 4-ethylidene-l-proline, enters the condensation reaction. The subsequent
postcondensation steps are initiated by the introduction of an additional
double bond catalyzed by a FAD-dependent oxidoreductase, which we
demonstrated with Orf7 from anthramycin biosynthesis. The resulting
double bond arrangement presumably represents a prerequisite for further
modifications of the APD moieties. Our study gives general insight
into the diversification of APD moieties of natural PBDs and provides
proof-of-principle for precursor directed and combinatorial biosynthesis
of new PBD-based antitumor compounds
Supporting infomation to the Figure 2.
Phylogenetic analysis of 17 putative ABC transporters from the isolated strains with 23 reference sequences of ABC transporters described in actinobacteria. An alignment of amino-acid ABC transporter sequences. The phylogenies inferred by Bayesian analysis, maximum-likelihood and neighbor-joining methods
Lincosamide Synthetase—A Unique Condensation System Combining Elements of Nonribosomal Peptide Synthetase and Mycothiol Metabolism
<div><p>In the biosynthesis of lincosamide antibiotics lincomycin and celesticetin, the amino acid and amino sugar units are linked by an amide bond. The respective condensing enzyme lincosamide synthetase (LS) is expected to be an unusual system combining nonribosomal peptide synthetase (NRPS) components with so far unknown amino sugar related activities. The biosynthetic gene cluster of celesticetin was sequenced and compared to the lincomycin one revealing putative LS coding ORFs shared in both clusters. Based on a bioassay and production profiles of <i>S. lincolnensis</i> strains with individually deleted putative LS coding genes, the proteins LmbC, D, E, F and V were assigned to LS function. Moreover, the newly recognized N-terminal domain of LmbN (LmbN-CP) was also assigned to LS as a NRPS carrier protein (CP). Surprisingly, the homologous CP coding sequence in celesticetin cluster is part of <i>ccbZ</i> gene adjacent to <i>ccbN</i>, the counterpart of <i>lmbN</i>, suggesting the gene rearrangement, evident also from still active internal translation start in <i>lmbN</i>, and indicating the direction of lincosamide biosynthesis evolution. The <i>in vitro</i> test with LmbN-CP, LmbC and the newly identified <i>S. lincolnensis</i> phosphopantetheinyl transferase Slp, confirmed the cooperation of the previously characterized NRPS A-domain LmbC with a <i>holo</i>-LmbN-CP in activation of a 4-propyl-L-proline precursor of lincomycin. This result completed the functional characterization of LS subunits resembling NRPS initiation module. Two of the four remaining putative LS subunits, LmbE/CcbE and LmbV/CcbV, exhibit low but significant homology to enzymes from the metabolism of mycothiol, the NRPS-independent system processing the amino sugar and amino acid units. The functions of particular LS subunits as well as cooperation of both NRPS-based and NRPS-independent LS blocks are discussed. The described condensing enzyme represents a unique hybrid system with overall composition quite dissimilar to any other known enzyme system.</p></div
Lincomycin Biosynthesis Involves a Tyrosine Hydroxylating Heme Protein of an Unusual Enzyme Family
<div><p>The gene lmbB2 of the lincomycin biosynthetic gene cluster of <i>Streptomyces lincolnensis</i> ATCC 25466 was shown to code for an unusual tyrosine hydroxylating enzyme involved in the biosynthetic pathway of this clinically important antibiotic. LmbB2 was expressed in <i>Escherichia coli</i>, purified near to homogeneity and shown to convert tyrosine to 3,4-dihydroxyphenylalanine (DOPA). In contrast to the well-known tyrosine hydroxylases (EC 1.14.16.2) and tyrosinases (EC 1.14.18.1), LmbB2 was identified as a heme protein. Mass spectrometry and Soret band-excited Raman spectroscopy of LmbB2 showed that LmbB2 contains heme b as prosthetic group. The CO-reduced differential absorption spectra of LmbB2 showed that the coordination of Fe was different from that of cytochrome P450 enzymes. LmbB2 exhibits sequence similarity to Orf13 of the anthramycin biosynthetic gene cluster, which has recently been classified as a heme peroxidase. Tyrosine hydroxylating activity of LmbB2 yielding DOPA in the presence of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH<sub>4</sub>) was also observed. Reaction mechanism of this unique heme peroxidases family is discussed. Also, tyrosine hydroxylation was confirmed as the first step of the amino acid branch of the lincomycin biosynthesis.</p></div
The lincomycin biosynthetic pathway.
<p>Gray background highlights the condensation step solved herein. Red—proteins with already proved functions, A—adenylation domain, CP—carrier protein, PPL—4-propyl-L-proline, MTL—methylthiolincosamide.</p
Structures and biosynthetic gene clusters of natural lincosamides.
<p>The resistance genes are marked in grey, the genes homologous in both gene clusters are black. The genes highlighted by red line exhibit inactivation pattern enabling their assignment to condensation reaction, i.e. formation of amide bond (in red oval in the structures).</p
Analysis of LmbB2 by CD spectroscopy.
<p>(A) The far-UV CD spectrum of LmbB2. (B) Thermal denaturation of LmbB2 in the presence of 0 µM (blue curve), 50 µM (red curve) and 250 µM (green curve) concentration of BH<sub>4</sub> bear witness of the stabilization of the LmbB2 structure by BH<sub>4</sub>.</p
Effect of BH<sub>4</sub> on melting temperature (<i>T</i><sub>m</sub>) of LmbB2.
<p>Effect of BH<sub>4</sub> on melting temperature (<i>T</i><sub>m</sub>) of LmbB2.</p
Abbreviations of specific compounds and proteins.
<p>Abbreviations of specific compounds and proteins.</p