Browse
Submit Data
Databases
API
Help

Dataset Information

19 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

SylC catalyzes ureido-bond formation during biosynthesis of the proteasome inhibitor syringolin A.

ABSTRACT: Syringolins are a class of cyclic tripeptide natural products that are potent and irreversible inhibitors of the eukaryotic proteasome. In addition to being hybrid NRPS/PKS molecules, they also feature an unusual ureido-linkage (red) between two amino acid monomers. Here we report the first in vitro characterization of enzymatic ureido-linkage formation which is catalyzed by an NRPS, SylC. Using (13)C- and (18)O-labeling studies, we show that biosynthesis occurs via N-carboxylation to form an initial N-carboxy-aminoacyl-S-Ppant enzyme intermediate which undergoes intramolecular cyclization followed by condensation with a second amino acid to form the ureido-containing dipeptide product.

SUBMITTER: Imker HJ

PROVIDER: S-EPMC2800832 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

SylC catalyzes ureido-bond formation during biosynthesis of the proteasome inhibitor syringolin A.

Imker Heidi J HJ Walsh Christopher T CT Wuest William M WM

Journal of the American Chemical Society 20091201 51

Syringolins are a class of cyclic tripeptide natural products that are potent and irreversible inhibitors of the eukaryotic proteasome. In addition to being hybrid NRPS/PKS molecules, they also feature an unusual ureido-linkage (red) between two amino acid monomers. Here we report the first in vitro characterization of enzymatic ureido-linkage formation which is catalyzed by an NRPS, SylC. Using (13)C- and (18)O-labeling studies, we show that biosynthesis occurs via N-carboxylation to form an in ...[more]

PMID: 19968303

Similar Datasets

Biosynthesis of the proteasome inhibitor syringolin A: the ureido group joining two amino acids originates from bicarbonate.

Project description:BackgroundSyringolin A, an important virulence factor in the interaction of the phytopathogenic bacterium Pseudomonas syringae pv. syringae B728a with its host plant Phaseolus vulgaris (bean), was recently shown to irreversibly inhibit eukaryotic proteasomes by a novel mechanism. Syringolin A is synthesized by a mixed non-ribosomal peptide synthetase/polyketide synthetase and consists of a tripeptide part including a twelve-membered ring with an N-terminal valine that is joined to a second valine via a very unusual ureido group. Analysis of sequence and architecture of the syringolin A synthetase gene cluster with the five open reading frames sylA-sylE allowed to formulate a biosynthesis model that explained all structural features of the tripeptide part of syringolin A but left the biosynthesis of the unusual ureido group unaccounted for.ResultsWe have cloned a 22 kb genomic fragment containing the sylA-sylE gene cluster but no other complete gene into the broad host range cosmid pLAFR3. Transfer of the recombinant cosmid into Pseudomonas putida and P. syringae pv. syringae SM was sufficient to direct the biosynthesis of bona fide syringolin A in these heterologous organisms whose genomes do not contain homologous genes. NMR analysis of syringolin A isolated from cultures grown in the presence of NaH(13)CO(3) revealed preferential (13)C-labeling at the ureido carbonyl position.ConclusionThe results show that no additional syringolin A-specific genes were needed for the biosynthesis of the enigmatic ureido group joining two amino acids. They reveal the source of the ureido carbonyl group to be bicarbonate/carbon dioxide, which we hypothesize is incorporated by carbamylation of valine mediated by the sylC gene product(s). A similar mechanism may also play a role in the biosynthesis of other ureido-group-containing NRPS products known largely from cyanobacteria.

| S-EPMC2773804 | biostudies-literature

Production of proteasome inhibitor syringolin A by the endophyte Rhizobium sp. strain AP16.

Project description:Syringolin A, the product of a mixed nonribosomal peptide synthetase/polyketide synthase encoded by the syl gene cluster, is a virulence factor secreted by certain Pseudomonas syringae strains. Together with the glidobactins produced by a number of beta- and gammaproteobacterial human and animal pathogens, it belongs to the syrbactins, a structurally novel class of proteasome inhibitors. In plants, proteasome inhibition by syringolin A-producing P. syringae strains leads to the suppression of host defense pathways requiring proteasome activity, such as the ones mediated by salicylic acid and jasmonic acid. Here we report the discovery of a syl-like gene cluster with some unusual features in the alphaproteobacterial endophyte Rhizobium sp. strain AP16 that encodes a putative syringolin A-like synthetase whose components share 55% to 65% sequence identity (72% to 79% similarity) at the amino acid level. As revealed by average nucleotide identity (ANI) calculations, this strain likely belongs to the same species as biocontrol strain R. rhizogenes K84 (formely known as Agrobacterium radiobacter K84), which, however, carries a nonfunctional deletion remnant of the syl-like gene cluster. Here we present a functional analysis of the syl-like gene cluster of Rhizobium sp. strain AP16 and demonstrate that this endophyte synthesizes syringolin A and some related minor variants, suggesting that proteasome inhibition by syrbactin production can be important not only for pathogens but also for endophytic bacteria in the interaction with their hosts.

| S-EPMC4054136 | biostudies-literature

Investigation of Amide Bond Formation during Dehydrophos Biosynthesis.

Project description:Dehydrophos is a tripeptide phosphonate antibiotic produced by Streptomyces luridus. Its biosynthetic pathway involves the use of aminoacyl-tRNA (aa-tRNA) for amide bond formation. The first amide bond during biosynthesis is formed by DhpH-C, a peptidyltransferase that utilizes Leu-tRNALeu. DhpH-C is a member of a burgeoning family of natural product biosynthetic enzymes that make use of aa-tRNA outside of canonical translation activities in the cell. Here, we used site-directed mutagenesis of both DhpH-C and tRNALeu to investigate the enzyme mechanism and substrate specificity, respectively, and analyzed the substrate scope for the production of a set of dipeptides. DhpH-C appears to recognize both the amino acyl group on the tRNA and the tRNA acceptor stem, and the enzyme can accept other hydrophobic residues, in addition to leucine. These results contribute to a better understanding of enzyme-aa-tRNA interactions and the growing exploration of aa-tRNA usage beyond translation.

| S-EPMC5856630 | biostudies-literature

Pseudomonas syringae pv. syringae uses proteasome inhibitor syringolin A to colonize from wound infection sites.

Project description:Infection of plants by bacterial leaf pathogens at wound sites is common in nature. Plants defend wound sites to prevent pathogen invasion, but several pathogens can overcome spatial restriction and enter leaf tissues. The molecular mechanisms used by pathogens to suppress containment at wound infection sites are poorly understood. Here, we studied Pseudomonas syringae strains causing brown spot on bean and blossom blight on pear. These strains exist as epiphytes that can cause disease upon wounding caused by hail, sand storms and frost. We demonstrate that these strains overcome spatial restriction at wound sites by producing syringolin A (SylA), a small molecule proteasome inhibitor. Consequently, SylA-producing strains are able to escape from primary infection sites and colonize adjacent tissues along the vasculature. We found that SylA diffuses from the primary infection site and suppresses acquired resistance in adjacent tissues by blocking signaling by the stress hormone salicylic acid (SA). Thus, SylA diffusion creates a zone of SA-insensitive tissue that is prepared for subsequent colonization. In addition, SylA promotes bacterial motility and suppresses immune responses at the primary infection site. These local immune responses do not affect bacterial growth and were weak compared to effector-triggered immunity. Thus, SylA facilitates colonization from wounding sites by increasing bacterial motility and suppressing SA signaling in adjacent tissues.

| S-EPMC3610659 | biostudies-literature

Enzyme inhibition by hydroamination: design and mechanism of a hybrid carmaphycin-syringolin enone proteasome inhibitor.

Project description:Hydroamination reactions involving the addition of an amine to an inactivated alkene are entropically prohibited and require strong chemical catalysts. While this synthetic process is efficient at generating substituted amines, there is no equivalent in small molecule-mediated enzyme inhibition. We report an unusual mechanism of proteasome inhibition that involves a hydroamination reaction of alkene derivatives of the epoxyketone natural product carmaphycin. We show that the carmaphycin enone first forms a hemiketal intermediate with the catalytic Thr1 residue of the proteasome before cyclization by an unanticipated intramolecular alkene hydroamination reaction, resulting in a stable six-membered morpholine ring. The carmaphycin enone electrophile, which does not undergo a 1,4-Michael addition as previously observed with vinyl sulfone and α,β-unsaturated amide-based inhibitors, is partially reversible and gives insight into the design of proteasome inhibitors for cancer chemotherapy.

| S-EPMC4103027 | biostudies-literature

A water-soluble DsbB variant that catalyzes disulfide-bond formation in vivo.

Project description:Escherichia coli DsbB is a transmembrane enzyme that catalyzes the reoxidation of the periplasmic oxidase DsbA by ubiquinone. Here, we sought to convert membrane-bound DsbB into a water-soluble biocatalyst by leveraging a previously described method for in vivo solubilization of integral membrane proteins (IMPs). When solubilized DsbB variants were coexpressed with an export-defective copy of DsbA in the cytoplasm of wild-type E. coli cells, artificial oxidation pathways were created that efficiently catalyzed de novo disulfide-bond formation in a range of substrate proteins, in a manner dependent on both DsbA and quinone. Hence, DsbB solubilization was achieved with preservation of both catalytic activity and substrate specificity. Moreover, given the generality of the solubilization technique, the results presented here should pave the way to unlocking the biocatalytic potential of other membrane-bound enzymes whose utility has been limited by poor stability of IMPs outside of their native lipid-bilayer context.

| S-EPMC5562517 | biostudies-literature

Enzymatic timing and tailoring of macrolactamization in syringolin biosynthesis.

Project description:The enzymatic activation of 3,4-dehydrolysine and subsequent formation of the 12-membered syringolin macrolactam were investigated. The timing of the desaturation was elucidated through the analysis of the initial adenylation domain of SylD. The SylD-TTE didomain was characterized and demonstrated to be the catalyst for formation of 12-membered macrocycles. When the SylD thioesterase domain was reacted with a family of acyclic CoA both natural and unnatural macrocycles were generated.

| S-EPMC3163001 | biostudies-literature

Graphene catalyzes the reversible formation of a C-C bond between two molecules.

Project description:Carbon deposits are well-known inhibitors of transition metal catalysts. In contrast to this undesirable behavior, here we show that epitaxial graphene grown on Ru(0001) promotes the reversible formation of a C-C bond between -CH2CN and 7,7,8,8-tetracyano-p-quinodimethane (TCNQ). The catalytic role of graphene is multifaceted: First, it allows for an efficient charge transfer between the surface and the reactants, thus favoring changes in carbon hybridization; second, it holds the reactants in place and makes them reactive. The reaction is fully reversible by injecting electrons with an STM tip on the empty molecular orbitals of the product. The making and breaking of the C-C bond is accompanied by the switching off and on of a Kondo resonance, so that the system can be viewed as a reversible magnetic switch controlled by a chemical reaction.

| S-EPMC6294602 | biostudies-other

Piper nigrum CYP719A37 Catalyzes the Decisive Methylenedioxy Bridge Formation in Piperine Biosynthesis.

Project description:Black pepper (Piper nigrum) is among the world's most popular spices. Its pungent principle, piperine, has already been identified 200 years ago, yet the biosynthesis of piperine in black pepper remains largely enigmatic. In this report we analyzed the characteristic methylenedioxy bridge formation of the aromatic part of piperine by a combination of RNA-sequencing, functional expression in yeast, and LC-MS based analysis of substrate and product profiles. We identified a single cytochrome P450 transcript, specifically expressed in black pepper immature fruits. The corresponding gene was functionally expressed in yeast (Saccharomyces cerevisiae) and characterized for substrate specificity with a series of putative aromatic precursors with an aromatic vanilloid structure. Methylenedioxy bridge formation was only detected when feruperic acid (5-(4-hydroxy-3-methoxyphenyl)-2,4-pentadienoic acid) was used as a substrate, and the corresponding product was identified as piperic acid. Two alternative precursors, ferulic acid and feruperine, were not accepted. Our data provide experimental evidence that formation of the piperine methylenedioxy bridge takes place in young black pepper fruits after a currently hypothetical chain elongation of ferulic acid and before the formation of the amide bond. The partially characterized enzyme was classified as CYP719A37 and is discussed in terms of specificity, storage, and phylogenetic origin of CYP719 catalyzed reactions in magnoliids and eudicots.

| S-EPMC7826766 | biostudies-literature

PEARL-catalyzed peptide bond formation after chain reversal during the biosynthesis of non-ribosomal peptides.

Project description:A subset of nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are encoded in their biosynthetic gene clusters (BGCs) with enzymes annotated as lantibiotic dehydratases. The functions of these putative lantibiotic dehydratases remain unknown. Here, we characterize an NRPS-PKS BGC with a putative lantibiotic dehydratase from the bacterium Stackebrandtia nassauensis (sna). Heterologous expression revealed several metabolites produced by the BGC, and the omission of selected biosynthetic enzymes revealed the biosynthetic sequence towards these compounds. The putative lantibiotic dehydratase catalyzes peptide bond formation that extends the peptide scaffold opposite to the NRPS and PKS biosynthetic direction. The condensation domain of the NRPS catalyzes the formation of a ureido group, and bioinformatics analysis revealed distinct active site residues of ureido-generating condensation (UreaC) domains. This work demonstrates that the annotated lantibiotic dehydratase serves as a separate amide bond-forming machinery in addition to the NRPS, and that the lantibiotic dehydratase enzyme family possesses diverse catalytic activities in the biosynthesis of both ribosomal and non-ribosomal natural products.

| S-EPMC10769383 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data