Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Synergistic actions of tailoring enzymes in pradimicin biosynthesis.

ABSTRACT: Three key tailoring enzymes in pradimicin biosynthesis: PdmJ, PdmW, and PdmN, were investigated. PdmW was characterized as the C-6 hydroxylase by structural characterization of the corresponding product, 6-hydroxy-G-2A. The efficiencies of the C-5 and C-6 hydroxylations, catalyzed respectively by PdmJ and PdmW, were low when they were expressed individually with the early biosynthetic enzymes that form G-2A. When these two cytochrome P450 enzymes were co-expressed, a dihydroxylated product, 5,6-dihydroxy-G-2A, was efficiently produced, indicating that these two enzymes work synergistically in pradimicin biosynthesis. Heterologously expressed PdmN in Streptomyces coelicolor CH999 converted G-2A to JX137a by ligating a unit of D-alanine to the carboxyl group. PdmN has relaxed substrate specificity toward both amino acid donors and acceptors. Through combinatorial biosynthesis, a series of new pradimicin analogues were produced.

SUBMITTER: Napan K

PROVIDER: S-EPMC4214279 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Synergistic actions of tailoring enzymes in pradimicin biosynthesis.

Napan Kandy K Zhang Shuwei S Morgan Whitney W Anderson Thomas T Takemoto Jon Y JY Zhan Jixun J

Chembiochem : a European journal of chemical biology 20140822 15

Three key tailoring enzymes in pradimicin biosynthesis: PdmJ, PdmW, and PdmN, were investigated. PdmW was characterized as the C-6 hydroxylase by structural characterization of the corresponding product, 6-hydroxy-G-2A. The efficiencies of the C-5 and C-6 hydroxylations, catalyzed respectively by PdmJ and PdmW, were low when they were expressed individually with the early biosynthetic enzymes that form G-2A. When these two cytochrome P450 enzymes were co-expressed, a dihydroxylated product, 5,6- ...[more]

PMID: 25155298

Similar Datasets

A key cytochrome P450 hydroxylase in pradimicin biosynthesis.

Project description:Pradimicins A-C (1-3) are a group of antifungal and antiviral polyketides from Actinomadura hibisca. The sugar moieties in pradimicins are required for their biological activities. Consequently, the 5-OH that is used for glycosylation plays a critical role in pradimicin biosynthesis. A cytochrome P450 monooxygenase gene, pdmJ, was amplified from the genomic DNA of A. hibisca and expressed in Escherichia coli BL21(DE3). PdmJ introduced a hydroxyl group to G-2A (4), a key pradimicin biosynthetic intermediate, at C-5 to form JX134 (5). A d-Ala-containing pradimicin analog, JX137a (6) was tested as an alternative substrate, but no product was detected by LC-MS, indicating that PdmJ has strict substrate specificity. Kinetic studies revealed a typical substrate inhibition of PdmJ activity. The optimal substrate concentration for the highest velocity is 115?M under the test conditions. Moreover, the conversion rate of 4 to 5 was reduced by the presence of 6, likely due to competitive inhibition. Coexpression of PdmJ and a glucose 1-dehydrogenase in E. coli BL21(DE3) provides an efficient method to produce the important intermediate 5 from 4.

| S-EPMC3248948 | biostudies-literature

Tailoring enzymes involved in the biosynthesis of angucyclines contain latent context-dependent catalytic activities.

Project description:Comparison of homologous angucycline modification enzymes from five closely related Streptomyces pathways (pga, cab, jad, urd, lan) allowed us to deduce the biosynthetic steps responsible for the three alternative outcomes: gaudimycin C, dehydrorabelomycin, and 11-deoxylandomycinone. The C-12b-hydroxylated urdamycin and gaudimycin metabolites appear to be the ancestral representatives from which landomycins and jadomysins have evolved as a result of functional divergence of the ketoreductase LanV and hydroxylase JadH, respectively. Specifically, LanV has acquired affinity for an earlier biosynthetic intermediate resulting in a switch in biosynthetic order and lack of hydroxyls at C-4a and C-12b, whereas in JadH, C-4a/C-12b dehydration has evolved into an independent secondary function replacing C-12b hydroxylation. Importantly, the study reveals that many of the modification enzymes carry several alternative, hidden, or ancestral catalytic functions, which are strictly dependent on the biosynthetic context.

| S-EPMC3361699 | biostudies-literature

Post-polyketide synthase steps in iso-migrastatin biosynthesis, featuring tailoring enzymes with broad substrate specificity.

Project description:The iso-migrastatin (iso-MGS) biosynthetic gene cluster from Streptomyces platensis NRRL 18993 consists of 11 genes, featuring an acyltransferase (AT)-less type I polyketide synthase (PKS) and three tailoring enzymes MgsIJK. Systematic inactivation of mgsIJK in S. platensis enabled us to (i) identify two nascent products of the iso-MGS AT-less type I PKS, establishing an unprecedented novel feature for AT-less type I PKSs, and (ii) account for the formation of all known post-PKS biosynthetic intermediates generated by the three tailoring enzymes MgsIJK, which possessed significant substrate promiscuities.

| S-EPMC3582021 | biostudies-literature

Three enzymes involved in the N-methylation and incorporation of the pradimicin sugar moieties.

Project description:Pradimicins are antifungal and antiviral natural products from Actinomadura hibisca P157-2. The sugar moieties play a critical role in the biological activities of these compounds. There are two glycosyltransferase genes in the pradimicin biosynthetic gene cluster, pdmS and pdmQ, which are putatively responsible for the introduction of the sugar moieties during pradimicin biosynthesis. In this study, we disrupted these two genes using a double crossover approach. Disruption of pdmS led to the production of pradimicinone I, the aglycon of pradimicin A, which confirmed that PdmS is the O-glycosyltransferase responsible for the first glycosylation step and attaching the 4',6'-dideoxy-4'-amino-d-galactose or 4',6'-dideoxy-4'-methylamino-d-galactose moiety to the 5-OH. Disruption of pdmQ resulted in the production of pradimicin B, indicating that this enzyme is the second glycosyltransferase that introduces the d-xylose moiety to the 3'-OH of the first sugar moiety. Insertion of an integrative plasmid before pdmO might have interfered with the dedicated promoter, yielding a mutant that produces pradimicin C as the major metabolite, which suggested that PdmO is the enzyme that specifically methylates the 4'-NH2 of the 4',6'-dideoxy-4'-amino-d-galactose moiety. Functional characterization of these sugar-decorating and -incorporating enzymes thus facilitates the understanding of the pradimicin biosynthetic pathway.

| S-EPMC4348331 | biostudies-literature

An acetyltransferase controls the metabolic flux in rubromycin polyketide biosynthesis by direct modulation of redox tailoring enzymes.

Project description:The often complex control of bacterial natural product biosynthesis typically involves global and pathway-specific transcriptional regulators of gene expression, which often limits the yield of bioactive compounds under laboratory conditions. However, little is known about regulation mechanisms on the enzymatic level. Here, we report a novel regulatory principle for natural products involving a dedicated acetyltransferase, which modifies a redox-tailoring enzyme and thereby enables pathway furcation and alternating pharmacophore assembly in rubromycin polyketide biosynthesis. The rubromycins such as griseorhodin (grh) A are complex bioactive aromatic polyketides from Actinobacteria with a hallmark bisbenzannulated [5,6]-spiroketal pharmacophore that is mainly installed by two flavoprotein monooxygenases. First, GrhO5 converts the advanced precursor collinone into the [6,6]-spiroketal containing dihydrolenticulone, before GrhO6 effectuates a ring contraction to afford the [5,6]-spiroketal. Our results show that pharmacophore assembly in addition involves the acetyl-CoA-dependent acetyltransferase GrhJ that activates GrhO6 to allow the rapid generation and release of its labile product, which is subsequently sequestered by ketoreductase GrhO10 and converted into a stable intermediate. Consequently, the biosynthesis is directed to the generation of canonical rubromycins, while the alternative spontaneous [5,6]-spiroketal hydrolysis to a ring-opened pathway product is thwarted. Presumably, this allows the bacteria to rapidly adjust the biosynthesis of functionally distinct secondary metabolites depending on nutrient and precursor (i.e. acetyl-CoA) availability. Our study thus illustrates how natural product biosynthesis can be enzymatically regulated and provides new perspectives for the improvement of in vitro enzyme activities and natural product titers via biotechnological approaches.

| S-EPMC9215129 | 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

Characterization and carbohydrate specificity of pradimicin S.

Project description:The pradimicin family of antibiotics is attracting attention due to its anti-infective properties and as a model for understanding the requirements for carbohydrate recognition by small molecules. Members of the pradimicin family are unique among natural products in their ability to bind sugars in a Ca(2+)-dependent manner, but the oligomerization to insoluble aggregates that occurs upon Ca(2+) binding has prevented detailed characterization of their carbohydrate specificity and biologically relevant form. Here we take advantage of the water solubility of pradimicin S (PRM-S), a sulfated glucose-containing analogue of pradimicin A (PRM-A), to show by NMR spectroscopy and analytical ultracentrifugation that at biologically relevant concentrations, PRM-S binds Ca(2+) to form a tetrameric species that selectively binds and engulfs the trisaccharide Man?1-3(Man?1-6)Man over mannose or mannobiose. In functional HIV-1 entry assays, IC(50) values of 2-4 ?M for PRM-S corrrelate with the concentrations at which oligomerization occurs as well as the affinities with which PRM-S binds the HIV surface envelope glycoprotein gp120. Together these data reveal the biologically active form of PRM-S, provide an explanation for previous speculations that PRM-A may contain a second mannose binding site, and expand our understanding of the characteristics that can engender a small molecule with the ability to function as a carbohydrate receptor.

| S-EPMC3422641 | biostudies-literature

Tailoring the P450 monooxygenase gene for FR-008/candicidin biosynthesis.

Project description:Tailoring steps are often important for the activity of mature antibiotics. Here, we report that novel decarboxylated FR-008/candicidin derivatives were obtained from the P450 monooxygenase gene fscP mutant of Streptomyces sp. strain FR-008. The toxicity of decarboxylated FR-008/candicidin derivatives has been shown to be greatly reduced compared to that of wild-type FR-008/candicidin.

| S-EPMC2655452 | biostudies-literature

Purine Biosynthesis Enzymes in Hippocampal Neurons.

Project description:Despite reports implicating disrupted purine metabolism in causing a wide spectrum of neurological defects, the mechanistic details of purine biosynthesis in neurons are largely unknown. As an initial step in filling that gap, we examined the expression and subcellular distribution of three purine biosynthesis enzymes (PFAS, PAICS and ATIC) in rat hippocampal neurons. Using immunoblotting and high-resolution light and electron microscopic analysis, we find that all three enzymes are broadly distributed in hippocampal neurons with pools of these enzymes associated with mitochondria. These findings suggest a potential link between purine metabolism and mitochondrial function in neurons and provide an impetus for further studies.

| S-EPMC6085884 | biostudies-literature

Subcellular localization of sterol biosynthesis enzymes.

Project description:Cholesterol synthesis is a complex, coordinated process involving a series of enzymes. As of today, our understanding of subcellular localization of cholesterol biosynthesis enzymes is far from complete. Considering the complexity and intricacies of this pathway and the importance of functions of DHCR7, DHCR24 and EBP enzymes for human health, we undertook a study to determine their subcellular localization and co-localization. Using expression constructs and antibody staining in cell cultures and transgenic mice, we found that all three enzymes are expressed in ER and nuclear envelope. However, their co-localization was considerably different across the cellular compartments. Furthermore, we observed that in the absence of DHCR7 protein, DHCR24 shows a compensatory upregulation in a Dhcr7-/- transgenic mouse model. The overall findings suggest that the sterol biosynthesis enzymes might not always work in a same functional complex, but that they potentially have different, multifunctional roles that go beyond the sterol biosynthesis pathway. Furthermore, the newly uncovered compensatory mechanism between DHCR7 and DHCR24 could be of importance for designing medications that would improve cholesterol production in patients with desmosterolosis and Smith-Lemli-Opitz syndrome.

| S-EPMC6467513 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

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