Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

In vitro kinetic analysis of substrate specificity in enterobactin biosynthetic lower pathway enzymes provides insight into the biochemical function of the hot dog-fold thioesterase EntH.

ABSTRACT: The Escherichia coli siderophore enterobactin is assembled from 2,3-dihydroxybenzoate (2,3-DHB) and l-serine by the nonribosomal peptide synthetases EntB and EntF. The processive thiol-template strategy used can be sabotaged by EntB misacylation. Through in vitro kinetic analysis, we demonstrate two potential routes to EntB misacylation and provide evidence for two mechanisms by which the hot dog-fold thioesterase EntH can potentially prevent or reverse EntB misacylation.

SUBMITTER: Chen D

PROVIDER: S-EPMC2929593 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

In vitro kinetic analysis of substrate specificity in enterobactin biosynthetic lower pathway enzymes provides insight into the biochemical function of the hot dog-fold thioesterase EntH.

Chen Danqi D Wu Rui R Bryan Tyrel L TL Dunaway-Mariano Debra D

Biochemistry 20090101 3

The Escherichia coli siderophore enterobactin is assembled from 2,3-dihydroxybenzoate (2,3-DHB) and l-serine by the nonribosomal peptide synthetases EntB and EntF. The processive thiol-template strategy used can be sabotaged by EntB misacylation. Through in vitro kinetic analysis, we demonstrate two potential routes to EntB misacylation and provide evidence for two mechanisms by which the hot dog-fold thioesterase EntH can potentially prevent or reverse EntB misacylation. ...[more]

PMID: 19119850

Similar Datasets

Structure and function of a Campylobacter jejuni thioesterase Cj0915, a hexameric hot dog fold enzyme.

Project description:Acyl-coenzyme A (CoA) thioesterases are a large family of enzymes that hydrolyze acyl-CoA esters to the free fatty acid and CoA and thereby regulate essential cellular functions such as lipid metabolism, membrane synthesis, signal transduction, and gene transcription. To better understand the virulence mechanisms of Campylobacter jejuni, and its possible link to membrane lipid biosynthesis, we have investigated C. jejuni thioesterases, annotated as putative proteins. While little is known about fatty acid biosynthesis and regulation in C. jejuni, remarkable differences in the genome and its organization from Escherichia coli, the paradigm system, raise questions as to the functions of these putative proteins. Here we present the crystal structure and biochemical analysis of Cj0915, defining the first functional thioesterase from C. jejuni. The structure of Cj0915 reveals the hot dog fold with an YciA-type hexameric assembly. Enzymatic assays performed with the purified protein show that Cj0915 is an efficient thioesterase with a broad specificity toward acyl-CoA substrates. This study provides a framework for investigation on roles of the Cj0915 thioesterase in virulence, and functional activities associated with the Cj0915 thioesterase in vivo.

| S-EPMC2683891 | biostudies-literature

The hotdog thioesterase EntH (YbdB) plays a role in vivo in optimal enterobactin biosynthesis by interacting with the ArCP domain of EntB.

Project description:In response to iron limitation, the siderophore enterobactin is synthesized and secreted by Escherichia coli. Its biosynthesis is performed by a series of enzymes encoded by the Ent gene cluster. Among the genes of this cluster, ybdB has not been implicated in enterobactin production to date. We demonstrate here an in vivo role for the hotdog protein EntH (YbdB) in the optimal production of enterobactin. Indeed, we showed that EntH is a thioesterase specifically produced under iron limitation conditions. Furthermore, EntH interacts specifically with the aryl carrier protein (ArCP) domain of EntB, a crucial bifunctional enzyme of the enterobactin biosynthesis pathway and a potential target of EntH thioesterase activity. A strain devoid of EntH is impaired for growth under iron limitation associated with the presence of the salicylate inhibitor, correlating with the diminution of enterobactin production under these conditions. Normal growth and enterobactin production are restored upon expression of entH in trans. Inversely, unnecessary overproduction of EntH provokes a fall of the quantity of siderophore produced under iron starvation conditions. Our findings point to a proofreading role for EntH during biosynthesis of enterobactin in vivo. EntH thioesterase activity could be required for cleaving wrongly charged molecules on the carrier protein EntB. This is the first description of such a role in the optimization of a nonribosomal biosynthesis pathway for a protein of the hotdog superfamily.

| S-EPMC2045218 | biostudies-literature

Structure of the bifunctional acyltransferase/decarboxylase LnmK from the leinamycin biosynthetic pathway revealing novel activity for a double-hot-dog fold.

Project description:The ?-branched C3 unit in leinamycin biosynthesis is installed by a set of four proteins, LnmFKLM. In vitro biochemical investigation confirmed that LnmK is a bifunctional acyltransferase/decarboxylase (AT/DC) that catalyzes first self-acylation using methylmalonyl-CoA as a substrate and subsequently transacylation of the methylmalonyl group to the phosphopantetheinyl group of the LnmL acyl carrier protein [Liu, T., Huang, Y., and Shen, B. (2009) J. Am. Chem. Soc. 131, 6900-6901]. LnmK shows no sequence homology to proteins of known function, representing a new family of AT/DC enzymes. Here we report the X-ray structure of LnmK. LnmK is homodimer with each of the monomers adopting a double-hot-dog fold. Cocrystallization of LnmK with methylmalonyl-CoA revealed an active site tunnel terminated by residues from the dimer interface. In contrast to canonical AT and ketosynthase enzymes that employ Ser or Cys as an active site residue, none of these residues are found in the vicinity of the LnmK active site. Instead, three tyrosines were identified, one of which, Tyr62, was established, by site-directed mutagenesis, to be the most likely active site residue for the AT activity of LnmK. LnmK represents the first AT enzyme that employs a Tyr as an active site residue and the first member of the family of double-hot-dog fold enzymes that displays an AT activity known to date. The LnmK structure sets the stage for probing of the DC activity of LnmK through site-directed mutagenesis. These findings highlight natural product biosynthetic machinery as a rich source of novel enzyme activities, mechanisms, and structures.

| S-EPMC3567400 | biostudies-literature

Analysis of proteins with the 'hot dog' fold: prediction of function and identification of catalytic residues of hypothetical proteins.

Project description:BackgroundThe hot dog fold has been found in more than sixty proteins since the first report of its existence about a decade ago. The fold appears to have a strong association with fatty acid biosynthesis, its regulation and metabolism, as the proteins with this fold are predominantly coenzyme A-binding enzymes with a variety of substrates located at their active sites.ResultsWe have analyzed the structural features and sequences of proteins having the hot dog fold. This study reveals that though the basic architecture of the fold is well conserved in these proteins, significant differences exist in their sequence, nature of substrate and oligomerization. Segments with certain conserved sequence motifs seem to play crucial structural and functional roles in various classes of these proteins.ConclusionThe analysis led to predictions regarding the functional classification and identification of possible catalytic residues of a number of hot dog fold-containing hypothetical proteins whose structures were determined in high throughput structural genomics projects.

| S-EPMC2698920 | biostudies-literature

Thioesterase superfamily member 2 (Them2)/acyl-CoA thioesterase 13 (Acot13): a homotetrameric hotdog fold thioesterase with selectivity for long-chain fatty acyl-CoAs.

Project description:Them2 (thioesterase superfamily member 2) is a 140-amino-acid protein of unknown biological function that comprises a single hotdog fold thioesterase domain. On the basis of its putative association with mitochondria, accentuated expression in oxidative tissues and interaction with StarD2 (also known as phosphatidylcholine-transfer protein, PC-TP), a regulator of fatty acid metabolism, we explored whether Them2 functions as a physiologically relevant fatty acyl-CoA thioesterase. In solution, Them2 formed a stable homotetramer, which denatured in a single transition at 59.3 degrees C. Them2 exhibited thioesterase activity for medium- and long-chain acyl-CoAs, with Km values that decreased exponentially as a function of increasing acyl chain length. Steady-state kinetic parameters for Them2 were characteristic of long-chain mammalian acyl-CoA thioesterases, with minimal values of Km and maximal values of kcat/Km observed for myristoyl-CoA and palmitoyl-CoA. For these acyl-CoAs, substrate inhibition was observed when concentrations approached their critical micellar concentrations. The acyl-CoA thioesterase activity of Them2 was optimized at physiological temperature, ionic strength and pH. For both myristoyl-CoA and palmitoyl-CoA, the addition of StarD2 increased the kcat of Them2. Enzymatic activity was decreased by the addition of phosphatidic acid/phosphatidylcholine small unilamellar vesicles. Them2 expression, which was most pronounced in mouse heart, was associated with mitochondria and was induced by activation of PPARalpha (peroxisome-proliferator-activated receptor alpha). We conclude that, under biological conditions, Them2 probably functions as a homotetrameric long-chain acyl-CoA thioesterase. Accordingly, Them2 has been designated as the 13th member of the mammalian acyl-CoA thioesterase family, Acot13.

| S-EPMC3086008 | biostudies-literature

Kinetic characterization of Cas9 enzymes.

Project description:Bacterial adaptive immune systems employ clustered regularly interspaced short palindromic repeats (CRISPR) along with their CRISPR-associated genes (Cas) to form CRISPR RNA (crRNA)-guided surveillance complexes, which target foreign nucleic acids for destruction. Cas9 is unique in that it is composed of a single polypeptide that utilizes both a crRNA and a trans-activating crRNA (tracrRNA) or a single guide RNA to create double-stranded breaks in sequences complementary to the RNA via the HNH and RuvC nuclease domains. Cas9 has become a revolutionary tool for gene-editing applications. Here, we describe methods for studying the cleavage activities of Cas9. We describe protocols for rapid quench-flow and stopped-flow kinetics and interpretation of the results. The protocols detailed here will be paramount for understanding the mechanistic basis for specificity of this enzyme, especially in efforts to improve accuracy for clinical use.

| S-EPMC8364395 | biostudies-literature

Biosynthetic enzymes of unusual microbial sugars.

Project description:The biological importance of proteins and nucleic acids in the natural world is undeniable, and research efforts on these macromolecules have often overshadowed those directed at carbohydrates. It is now known, however, that carbohydrates not only play roles in energy storage and plant cell wall structure, but are also intimately involved in such processes as fertilization, the immune response, and cell adhesion. Indeed, recent years have seen an explosion in research efforts directed at uncovering and understanding new sugar moieties. The dideoxysugars and trideoxysugars, which are synthesized by a variety of bacteria, fungi, and plants, represent an especially intriguing class of carbohydrates. They are found, for example, on the lipopolysaccharides of some Gram-negative bacteria or on antibacterial agents such as erythromycin. Many of them are formed from simple monosaccharides such as glucose-6-phosphate or fructose-6-phosphate via a myriad of enzymatic reactions including acetylations, aminations, dehydrations, epimerizations, reductions, and methylations. In this review we focus on the recent structural investigations of the bacterial N-acetyltransferases and the PLP-dependent aminotransferases that function on nucleotide-linked sugar substrates.

| S-EPMC4254661 | biostudies-literature

Mechanistic Understanding of Lanthipeptide Biosynthetic Enzymes.

Project description:Lanthipeptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) that display a wide variety of biological activities, from antimicrobial to antiallodynic. Lanthipeptides that display antimicrobial activity are called lantibiotics. The post-translational modification reactions of lanthipeptides include dehydration of Ser and Thr residues to dehydroalanine and dehydrobutyrine, a transformation that is carried out in three unique ways in different classes of lanthipeptides. In a cyclization process, Cys residues then attack the dehydrated residues to generate the lanthionine and methyllanthionine thioether cross-linked amino acids from which lanthipeptides derive their name. The resulting polycyclic peptides have constrained conformations that confer their biological activities. After installation of the characteristic thioether cross-links, tailoring enzymes introduce additional post-translational modifications that are unique to each lanthipeptide and that fine-tune their activities and/or stability. This review focuses on studies published over the past decade that have provided much insight into the mechanisms of the enzymes that carry out the post-translational modifications.

| S-EPMC5408752 | biostudies-literature

Leukotriene biosynthetic enzymes as therapeutic targets.

Project description:Leukotrienes are powerful immune-regulating lipid mediators with established pathogenic roles in inflammatory allergic diseases of the respiratory tract - in particular, asthma and hay fever. More recent work indicates that these lipids also contribute to low-grade inflammation, a hallmark of cardiovascular, neurodegenerative, and metabolic diseases as well as cancer. Biosynthesis of leukotrienes involves oxidative metabolism of arachidonic acid and proceeds via a set of soluble and membrane enzymes that are primarily expressed by cells of myeloid origin. In activated immune cells, these enzymes assemble at the endoplasmic and perinuclear membrane, constituting a biosynthetic complex. This Review describes recent advances in our understanding of the components of the leukotriene-synthesizing enzyme machinery, emerging opportunities for pharmacological intervention, and the development of new medicines exploiting both antiinflammatory and pro-resolving mechanisms.

| S-EPMC6026001 | biostudies-literature

Structure and evolution of ENTH and VHS/ENTH-like domains in tepsin.

Project description:Tepsin is currently the only accessory trafficking protein identified in adaptor-related protein 4 (AP4)-coated vesicles originating at the trans-Golgi network (TGN). The molecular basis for interactions between AP4 subunits and motifs in the tepsin C-terminus have been characterized, but the biological role of tepsin remains unknown. We determined X-ray crystal structures of the tepsin epsin N-terminal homology (ENTH) and VHS/ENTH-like domains. Our data reveal unexpected structural features that suggest key functional differences between these and similar domains in other trafficking proteins. The tepsin ENTH domain lacks helix0, helix8 and a lipid binding pocket found in epsin1/2/3. These results explain why tepsin requires AP4 for its membrane recruitment and further suggest ENTH domains cannot be defined solely as lipid binding modules. The VHS domain lacks helix8 and thus contains fewer helices than other VHS domains. Structural data explain biochemical and biophysical evidence that tepsin VHS does not mediate known VHS functions, including recognition of dileucine-based cargo motifs or ubiquitin. Structural comparisons indicate the domains are very similar to each other, and phylogenetic analysis reveals their evolutionary pattern within the domain superfamily. Phylogenetics and comparative genomics further show tepsin within a monophyletic clade that diverged away from epsins early in evolutionary history (~1500 million years ago). Together, these data provide the first detailed molecular view of tepsin and suggest tepsin structure and function diverged away from other epsins. More broadly, these data highlight the challenges inherent in classifying and understanding protein function based only on sequence and structure.

| S-EPMC5567745 | 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