Browse
Submit Data
Databases
API
Help

Dataset Information

7 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

p-Nitrophenyl esters provide new insights and applications for the thiolase enzyme OleA.

ABSTRACT: The OleA enzyme is distinct amongst thiolase enzymes in binding two long (≥C₈) acyl chains into structurally-opposed hydrophobic channels, denoted A and B, to carry out a non-decarboxylative Claisen condensation reaction and initiate the biosynthesis of membrane hydrocarbons and β-lactone natural products. OleA has now been identified in hundreds of diverse bacteria via bioinformatics and high-throughput screening using p-nitrophenyl alkanoate esters as surrogate substrates. In the present study, p-nitrophenyl esters were used to probe the reaction mechanism of OleA and shown to be incorporated into Claisen condensation products for the first time. p-Nitrophenyl alkanoate substrates alone were shown not to undergo Claisen condensation, but co-incubation of p-nitrophenyl esters and CoA thioesters produced mixed Claisen products. Mixed product reactions were shown to initiate via acyl group transfer from a p-nitrophenyl carrier to the enzyme active site cysteine, C143. Acyl chains esterified to p-nitrophenol were synthesized and shown to undergo Claisen condensation with an acyl-CoA substrate, showing potential to greatly expand the range of possible Claisen products. Using p-nitrophenyl 1-¹³C-decanoate, the Channel A bound thioester chain was shown to act as the Claisen nucleophile, representing the first direct evidence for the directionality of the Claisen reaction in any OleA enzyme. These results both provide new insights into OleA catalysis and open a path for making unnatural hydrocarbon and β-lactone natural products for biotechnological applications using cheap and easily synthesized p-nitrophenyl esters.

SUBMITTER: Smith MD

PROVIDER: S-EPMC8180931 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

4-Nitrophenyl activated esters are superior synthons for indirect radiofluorination of biomolecules.

Project description:Indirect radiolabelling has for a long time been the mainstay strategy for radiofluorination of biomolecules. Acylation of biomolecules through the use of an 18F-labelled activated ester is a standard method for indirect radiolabelling. However, the preparation of 18F-labelled activated esters is typically a complex and multistep procedure. Herein, we describe the use of 4-nitrophenyl (PNP) activated esters to rapidly prepare 18F-labelled acylation synthons in one step. Furthermore, we present a comparative study of PNP activated esters and the commonly utilised 2,3,5,6-tetrafluorphenyl (TFP) activated esters under direct radiofluorination conditions and demonstrate their relative acylation behaviour. We demonstrate the superiority of PNP esters under direct radiofluorination conditions with favourable acylation kinetics.

| S-EPMC7517343 | biostudies-literature

Substrate Trapping in Crystals of the Thiolase OleA Identifies Three Channels That Enable Long Chain Olefin Biosynthesis.

Project description:Phylogenetically diverse microbes that produce long chain, olefinic hydrocarbons have received much attention as possible sources of renewable energy biocatalysts. One enzyme that is critical for this process is OleA, a thiolase superfamily enzyme that condenses two fatty acyl-CoA substrates to produce a ?-ketoacid product and initiates the biosynthesis of long chain olefins in bacteria. Thiolases typically utilize a ping-pong mechanism centered on an active site cysteine residue. Reaction with the first substrate produces a covalent cysteine-thioester tethered acyl group that is transferred to the second substrate through formation of a carbon-carbon bond. Although the basics of thiolase chemistry are precedented, the mechanism by which OleA accommodates two substrates with extended carbon chains and a coenzyme moiety-unusual for a thiolase-are unknown. Gaining insights into this process could enable manipulation of the system for large scale olefin production with hydrocarbon chains lengths equivalent to those of fossil fuels. In this study, mutagenesis of the active site cysteine in Xanthomonas campestris OleA (Cys143) enabled trapping of two catalytically relevant species in crystals. In the resulting structures, long chain alkyl groups (C12 and C14) and phosphopantetheinate define three substrate channels in a T-shaped configuration, explaining how OleA coordinates its two substrates and product. The C143A OleA co-crystal structure possesses a single bound acyl-CoA representing the Michaelis complex with the first substrate, whereas the C143S co-crystal structure contains both acyl-CoA and fatty acid, defining how a second substrate binds to the acyl-enzyme intermediate. An active site glutamate (Glu?117) is positioned to deprotonate bound acyl-CoA and initiate carbon-carbon bond formation.

| S-EPMC5207179 | biostudies-literature

Rational design of thiolase substrate specificity for metabolic engineering applications.

Project description:Metabolic engineering efforts require enzymes that are both highly active and specific toward the synthesis of a desired output product to be commercially feasible. The 3-hydroxyacid (3HA) pathway, also known as the reverse ?-oxidation or coenzyme-A-dependent chain-elongation pathway, can allow for the synthesis of dozens of useful compounds of various chain lengths and functionalities. However, this pathway suffers from byproduct formation, which lowers the yields of the desired longer chain products, as well as increases downstream separation costs. The thiolase enzyme catalyzes the first reaction in this pathway, and its substrate specificity at each of its two catalytic steps sets the chain length and composition of the chemical scaffold upon which the other downstream enzymes act. However, there have been few attempts reported in the literature to rationally engineer thiolase substrate specificity. In this study, we present a model-guided, rational design study of ordered substrate binding applied to two biosynthetic thiolases, with the goal of increasing the ratio of C6/C4 products formed by the 3HA pathway, 3-hydroxy-hexanoic acid and 3-hydroxybutyric acid. We identify thiolase mutants that result in nearly 10-fold increases in C6/C4 selectivity. Our findings can extend to other pathways that employ the thiolase for chain elongation, as well as expand our knowledge of sequence-structure-function relationship for this important class of enzymes.

| S-EPMC6131064 | biostudies-literature

Morphological studies of rose prickles provide new insights.

Project description:Prickles are common structures in plants that play a key role in defense against herbivores. In the Rosa genus, prickles are widely present with great diversity in terms of form and density. For cut rose production, prickles represent an important issue, as they can damage the flower and injure workers. Our objectives were to precisely describe the types of prickles that exist in roses, their tissues of origin and their development. We performed a detailed histological analysis of prickle initiation and development in a rose F1 population. Based on the prickle investigation of 110 roses, we proposed the first categorization of prickles in the Rosa genus. They are mainly divided into two categories, nonglandular prickles (NGPs) and glandular prickles (GPs), and subcategories were defined based on the presence/absence of hairs and branches. We demonstrated that NGPs and GPs both originate from multiple cells of the ground meristem beneath the protoderm. For GPs, the gland cells originate from the protoderm of the GP at the early developmental stage. Our findings clearly demonstrate that prickles are not modified trichomes (which originate from the protoderm). These conclusions are different from the current mainstream hypothesis. These results provide a foundation for further studies on prickle initiation and development in plants.

| S-EPMC8460668 | biostudies-literature

Genetic modifications of critical regulators provide new insights into regulation modes of raw-starch-digesting enzyme expression in Penicillium.

Project description:BackgroundStarch is a very abundant and renewable carbohydrate and an important feedstock for industrial applications. However, most starch-based products are not cost-efficient due to the high energy input needed in traditional enzymatic starch conversion processes. Raw-starch-digesting enzymes (RSDEs) from filamentous fungi have great commercial value in starch processing. However, the regulatory mechanisms associated with their production in filamentous fungi remain unknown.ResultsIn this study, we reported the novel finding that cellulolytic fungus Penicillium oxalicum 114-2 has broad RSDE activity. Four regulators, including the amylase transcription activator AmyR, the catabolite repression repressor CreA, the group III G protein α subunit PGA3, and the nonhistone chromosomal protein HepA, have been found to play a crucial regulatory role in RSDE expression. Enzymatic assays revealed that RSDE production significantly increased after the overexpression of AmyR and HepA, the deletion of CreA and the dominant activation of PGA3. RT-qPCR analysis demonstrated that there is a mutual regulation mode between the four regulators, and then formed a cascade regulation mechanism that is involved in RSDE expression. Comparative transcriptomic analysis between the wild-type strain and genetically engineered strains revealed differentially expressed genes that may mediate the RSDE expression.ConclusionsThe four different types of regulators were systematically investigated and found to form a regulatory network controlling RSDE gene expression. Our results provide a new insight into the regulatory mechanism of fungal amylolytic enzyme expression and offer a theoretical basis to rationally improve the RSDE yield in the future.

| S-EPMC9158223 | biostudies-literature

Genome sequencing and analysis of Talaromyces pinophilus provide insights into biotechnological applications.

Project description:Species from the genus Talaromyces produce useful biomass-degrading enzymes and secondary metabolites. However, these enzymes and secondary metabolites are still poorly understood and have not been explored in depth because of a lack of comprehensive genetic information. Here, we report a 36.51-megabase genome assembly of Talaromyces pinophilus strain 1-95, with coverage of nine scaffolds of eight chromosomes with telomeric repeats at their ends and circular mitochondrial DNA. In total, 13,472 protein-coding genes were predicted. Of these, 803 were annotated to encode enzymes that act on carbohydrates, including 39 cellulose-degrading and 24 starch-degrading enzymes. In addition, 68 secondary metabolism gene clusters were identified, mainly including T1 polyketide synthase genes and nonribosomal peptide synthase genes. Comparative genomic analyses revealed that T. pinophilus 1-95 harbors more biomass-degrading enzymes and secondary metabolites than other related filamentous fungi. The prediction of the T. pinophilus 1-95 secretome indicated that approximately 50% of the biomass-degrading enzymes are secreted into the extracellular environment. These results expanded our genetic knowledge of the biomass-degrading enzyme system of T. pinophilus and its biosynthesis of secondary metabolites, facilitating the cultivation of T. pinophilus for high production of useful products.

| S-EPMC5428652 | biostudies-literature

New model systems provide insights into Myc-induced transformation.

Project description:The ability of Myc to promote cellular transformation is well established; however, a better understanding of the mechanisms through which Myc mediates tumorigenesis is essential for the development of therapeutic approaches to target this potent oncoprotein. Structure-function studies in rodent fibroblast cells have provided the basis for much of our current understanding of these mechanisms. To build on these approaches, we have characterized three novel human cell line models of Myc-dependent transformation: MCF10A, SH-EP Tet21/N-Myc, and LF1/TERT/LT/ST cells. We have also evaluated Myc family proteins (c-Myc and L-Myc), a naturally occurring isoform of Myc (MycS), and a set of N-terminal domain mutants (?MBII, W135E, T58A) for their ability to promote anchorage-independent growth in these models. Taken together, these results provide the field with three new human cell-based models to study Myc activity, highlight the importance of cellular context, and challenge the paradigm that the ability of Myc to promote tumorigenesis is exclusively MBII-dependent.

| S-EPMC3765156 | biostudies-literature

New epitopes in ovalbumin provide insights for cancer neoepitopes.

Project description:MHC I-restricted epitopes of chicken ovalbumin (OVA) were originally identified using CD8 T cells as probes. Here, using bioinformatics tools, we identify four additional epitopes in OVA in addition to a cryptic epitope. Each new epitope is presented in vivo, as deduced from the lack of CD8 response to it in OVA-transgenic mice. In addition, CD8 responses to the known and novel epitopes are examined in C57BL/6 mice exposed to the OVA-expressing tumor E.G7 in several ways. No responses to any epitope including SIINFEKL are detected in mice with growing E.G7 or mice immunized with the tumor. Only in E.G7-bearing mice treated with an anti-CTLA4 antibody which depletes tumor-infiltrating regulatory T cells, CD8 responses to SIINFEKL and the novel epitope EKYNLTSVL are detected. Finally, all epitopes fails to treat mice with pre-existing tumors. These observations force an important re-consideration of the common assumptions about the therapeutic value of neoepitopes detected by CD8 responses in tumor-bearing hosts.

| S-EPMC6538383 | biostudies-literature

Crystal structures of N-acetylmannosamine kinase provide insights into enzyme activity and inhibition.

Project description:Sialic acids are essential components of membrane glycoconjugates. They are responsible for the interaction, structure, and functionality of all deuterostome cells and have major functions in cellular processes in health and diseases. The key enzyme of the biosynthesis of sialic acid is the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase that transforms UDP-N-acetylglucosamine to N-acetylmannosamine (ManNAc) followed by its phosphorylation to ManNAc 6-phosphate and has a direct impact on the sialylation of cell surface components. Here, we present the crystal structures of the human N-acetylmannosamine kinase (MNK) domain of UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase in complexes with ManNAc at 1.64 Å resolution, MNK·ManNAc·ADP (1.82 Å) and MNK·ManNAc 6-phosphate · ADP (2.10 Å). Our findings offer detailed insights in the active center of MNK and serve as a structural basis to design inhibitors. We synthesized a novel inhibitor, 6-O-acetyl-ManNAc, which is more potent than those previously tested. Specific inhibitors of sialic acid biosynthesis may serve to further study biological functions of sialic acid.

| S-EPMC3340156 | biostudies-literature

Structures of mouse DUOX1-DUOXA1 provide mechanistic insights into enzyme activation and regulation.

Project description:DUOX1, an NADPH oxidase family member, catalyzes the production of hydrogen peroxide. DUOX1 is expressed in various tissues, including the thyroid and respiratory tract, and plays a crucial role in processes such as thyroid hormone biosynthesis and innate host defense. DUOX1 co-assembles with its maturation factor DUOXA1 to form an active enzyme complex. However, the molecular mechanisms for activation and regulation of DUOX1 remain mostly unclear. Here, I present cryo-EM structures of the mammalian DUOX1-DUOXA1 complex, in the absence and presence of substrate NADPH, as well as DUOX1-DUOXA1 in an unexpected dimer-of-dimers configuration. These structures reveal atomic details of the DUOX1-DUOXA1 interaction, a lipid-mediated NADPH-binding pocket and the electron transfer path. Furthermore, biochemical and structural analyses indicate that the dimer-of-dimers configuration represents an inactive state of DUOX1-DUOXA1, suggesting an oligomerization-dependent regulatory mechanism. Together, my work provides structural bases for DUOX1-DUOXA1 activation and regulation.

| S-EPMC7644671 | 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