Browse
Submit Data
Databases
API
Help

Dataset Information

19 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Engineering P450 TamI as an Iterative Biocatalyst for Selective Late-Stage C-H Functionalization and Epoxidation of Tirandamycin Antibiotics.

ABSTRACT: Iterative P450 enzymes are powerful biocatalysts for selective late-stage C-H oxidation of complex natural product scaffolds. These enzymes represent useful tools for selectivity and cascade reactions, facilitating direct access to core structure diversification. Recently, we reported the structure of the multifunctional bacterial P450 TamI and elucidated the molecular basis of its substrate binding and strict reaction sequence at distinct carbon atoms of the substrate. Here, we report the design and characterization of a toolbox of TamI biocatalysts, generated by mutations at Leu101, Leu244, and/or Leu295, that alter the native selectivity, step sequence, and number of reactions catalyzed, including the engineering of a variant capable of catalyzing a four-step oxidative cascade without the assistance of the flavoprotein and oxidative partner TamL. The tuned enzymes override inherent substrate reactivity, enabling catalyst-controlled C-H functionalization and alkene epoxidation of the tetramic acid-containing natural product tirandamycin. Five bioactive tirandamycin derivatives (6-10) were generated through TamI-mediated enzymatic synthesis. Quantum mechanics calculations and MD simulations provide important insights into the basis of altered selectivity and underlying biocatalytic mechanisms for enhanced continuous oxidation of the iterative P450 TamI.

SUBMITTER: Espinoza RV

PROVIDER: S-EPMC8739753 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Function and Structure of MalA/MalA', Iterative Halogenases for Late-Stage C-H Functionalization of Indole Alkaloids.

Project description:Malbrancheamide is a dichlorinated fungal indole alkaloid isolated from both Malbranchea aurantiaca and Malbranchea graminicola that belongs to a family of natural products containing a characteristic bicyclo[2.2.2]diazaoctane core. The introduction of chlorine atoms on the indole ring of malbrancheamide differentiates it from other members of this family and contributes significantly to its biological activity. In this study, we characterized the two flavin-dependent halogenases involved in the late-stage halogenation of malbrancheamide in two different fungal strains. MalA and MalA' catalyze the iterative dichlorination and monobromination of the free substrate premalbrancheamide as the final steps in the malbrancheamide biosynthetic pathway. Two unnatural bromo-chloro-malbrancheamide analogues were generated through MalA-mediated chemoenzymatic synthesis. Structural analysis and computational studies of MalA' in complex with three substrates revealed that the enzyme represents a new class of zinc-binding flavin-dependent halogenases and provides new insights into a potentially unique reaction mechanism.

| S-EPMC5595095 | biostudies-literature

Molecular Basis of Iterative C─H Oxidation by TamI, a Multifunctional P450 monooxygenase from the Tirandamycin Biosynthetic Pathway.

Project description:Biocatalysis offers an expanding and powerful strategy to construct and diversify complex molecules by C─H bond functionalization. Due to their high selectivity, enzymes have become an essential tool for C─H bond functionalization and offer complementary reactivity to small-molecule catalysts. Hemoproteins, particularly cytochromes P450, have proven effective for selective oxidation of unactivated C─H bonds. Previously, we reported the in vitro characterization of an oxidative tailoring cascade in which TamI, a multifunctional P450 functions co-dependently with the TamL flavoprotein to catalyze regio- and stereoselective hydroxylations and epoxidation to yield tirandamycin A and tirandamycin B. TamI follows a defined order including 1) C10 hydroxylation, 2) C11/C12 epoxidation, and 3) C18 hydroxylation. Here we present a structural, biochemical, and computational investigation of TamI to understand the molecular basis of its substrate binding, diverse reactivity, and specific reaction sequence. The crystal structure of TamI in complex with tirandamycin C together with molecular dynamics simulations and targeted mutagenesis suggest that hydrophobic interactions with the polyene chain of its natural substrate are critical for molecular recognition. QM calculations and molecular dynamics simulations of TamI with variant substrates provided detailed information on the molecular basis of sequential reactivity, and pattern of regio- and stereo-selectivity in catalyzing the three-step oxidative cascade.

| S-EPMC7869845 | biostudies-literature

Discovery of potent parthenolide-based antileukemic agents enabled by late-stage P450-mediated C-H functionalization.

Project description:The sesquiterpene lactone parthenolide has recently attracted considerable attention owing to its promising antitumor properties, in particular in the context of stem-cell cancers including leukemia. Yet, the lack of viable synthetic routes for re-elaborating this complex natural product has represented a fundamental obstacle toward further optimization of its pharmacological properties. Here, we demonstrate how this challenge could be addressed via selective, late-stage sp(3) C-H bond functionalization mediated by P450 catalysts with tailored site-selectivity. Taking advantage of our recently introduced tools for high-throughput P450 fingerprinting and fingerprint-driven P450 reactivity prediction, we evolved P450 variants useful for carrying out the highly regioselective hydroxylation of two aliphatic sites (C9 and C14) in parthenolide carbocyclic backbone. By chemoenzymatic synthesis, a panel of novel C9- and C14-modified parthenolide analogs were generated in order to gain initial structure-activity insights on these previously inaccessible sites of the molecule. Notably, some of these compounds were found to possess significantly improved antileukemic potency against primary acute myeloid leukemia cells, while exhibiting low toxicity against normal mature and progenitor hematopoietic cells. By identifying two 'hot spots' for improving the anticancer properties of parthenolide, this study highlights the potential of P450-mediated C-H functionalization as an enabling, new strategy for the late-stage manipulation of bioactive natural product scaffolds.

| S-EPMC3904131 | biostudies-literature

Cytochrome P450 oxidase SlgO1 catalyzes the biotransformation of tirandamycin C to a new tirandamycin derivative.

Project description:In the present study, an Escherichia coli whole cell system with overexpression of a cytochrome P450 oxidase SlgO1 involved in streptolydigin biosynthetic pathway, an E. coli flavodoxin NADP+ oxidoreductase (EcFLDR), and an E. coli flavodoxin A (EcFLDA) were constructed. Biotransformation experiments revealed that SlgO1 can convert tirandamycin C to tirandamycin F, indicating that it can introduce a hydroxyl group into the C-10 position of tirandamycin C. Subsequently, slgO1 was cloned into pSET152AKE vector under the downstream of ermE* promoter, which was, respectively, introduced into Streptomyces sp. SCSIO1666 (tirandamycin B producer), Streptomyces sp. Ju1008 (tirandamycin C producer), and Streptomyces sp. Ju1009 (tirandamycin E producer). A novel tirandamycin derivative tirandamycin L accumulated in the engineered strain Streptomyces sp. Ju1008::slgO1 was isolated and its structure was determined on the basis of nuclear magnetic resonance (NMR) and mass spectrometry. Unlike most of the identified tirandamycins, tirandamycin L possessed a rare C-11-C-12 saturated bond as well as a C-10 ketone moiety. In addition, tirandamycin L showed weaker antibacterial activity. Based on the structure of tirandamycin L, SlgO1 was proposed to be responsible for multiple modifications toward tirandamycin C, including the formation of C-10 hydroxyl and C-11-C-12 saturated bond.

| S-EPMC6368906 | biostudies-literature

SuFEx Click Chemistry Enabled Late-Stage Drug Functionalization.

Project description:Sulfur(VI) Fluoride Exchange (SuFEx) is a new family of click chemistry transformations which relies on readily available materials to produce compounds bearing the SVI-F motif. The potential of SuFEx in drug discovery has just started to be explored. We report the first method of SuFEx chemistry for the conversion of phenolic compounds to their respective arylfluorosulfate derivatives in situ in 96-well plates. This method is compatible with automated synthesis and screening to quickly assess the biological activities of the in situ generated, crude products. Using this method, we perform late-stage functionalization of a panel of known anticancer drugs to generate the corresponding arylfluorosulfates. These in situ generated arylfluorosulfates are directly tested in a cancer-cell growth inhibition assay in parallel with their phenolic precursors. We discover three arylfluorosulfates that exhibit improved anticancer cell proliferation activities compared to their phenol precursors. Among these three compounds, the fluorosulfate derivative of Fulvestrant possesses significantly enhanced activity to down-regulate estrogen receptor (ER) expression in ER+ breast cancer cell line MCF-7 and the fluorosulfate derivative of Combretastatin A4-a general anticancer drug currently being evaluated under clinical trials-exhibits a 70-fold increase in potency in the drug resistant colon cancer cell line HT-29.

| S-EPMC6192542 | biostudies-literature

Aqueous Benzylic C-H Trifluoromethylation for Late-Stage Functionalization.

Project description:The installation of trifluoromethyl groups has become an essential step across a number of industries such as agrochemicals, drug discovery, and materials. Consequently, the rapid introduction of this critical functional group in a predictable fashion would benefit current practitioners in those fields. This communication describes a mild trifluoromethylation of benzylic C-H bonds with high selectivity for the least hindered hydrogen atom. The reaction provides monotrifluoromethylation and proceeds in an environmentally friendly acetone/water solvent system. The method can be used to install benzylic trifluoromethyl groups on highly functionalized drug molecules.

| S-EPMC6310231 | biostudies-literature

Chemoenzymatic Total Synthesis and Structural Diversification of Tylactone-Based Macrolide Antibiotics through Late-Stage Polyketide Assembly, Tailoring, and C-H Functionalization.

Project description:Polyketide synthases (PKSs) represent a powerful catalytic platform capable of effecting multiple carbon-carbon bond forming reactions and oxidation state adjustments. We explored the functionality of two terminal PKS modules that produce the 16-membered tylosin macrocycle, using them as biocatalysts in the chemoenzymatic synthesis of tylactone and its subsequent elaboration to complete the first total synthesis of the juvenimicin, M-4365, and rosamicin classes of macrolide antibiotics via late-stage diversification. Synthetic chemistry was employed to generate the tylactone hexaketide chain elongation intermediate that was accepted by the juvenimicin (Juv) ketosynthase of the penultimate JuvEIV PKS module. The hexaketide is processed through two complete modules (JuvEIV and JuvEV) in vitro, which catalyze elongation and functionalization of two ketide units followed by cyclization of the resulting octaketide into tylactone. After macrolactonization, a combination of in vivo glycosylation, selective in vitro cytochrome P450-mediated oxidation, and chemical oxidation was used to complete the scalable construction of a series of macrolide natural products in as few as 15 linear steps (21 total) with an overall yield of 4.6%.

| S-EPMC5532807 | biostudies-literature

Enabling highly (<i>R</i>)-enantioselective epoxidation of styrene by engineering unique non-natural P450 peroxygenases.

Project description:Unlike the excellent (S)-enantioselective epoxidation of styrene performed by natural styrene monooxygenases (ee > 99%), the (R)-enantioselective epoxidation of styrene has not yet achieved a comparable efficiency using natural or engineered oxidative enzymes. This report describes the H2O2-dependent (R)-enantioselective epoxidation of unfunctionalized styrene and its derivatives by site-mutated variants of a unique non-natural P450BM3 peroxygenase, working in tandem with a dual-functional small molecule (DFSM). The observed (R)-enantiomeric excess (ee) of styrene epoxidation is up to 99% with a turnover number (TON) of 918 by the best enantioselective mutant F87A/T268I/L181Q, while the best active mutant F87A/T268I/V78A/A184L (with 98% ee) gave a catalytic TON of 4350, representing the best activity of a P450 peroxygenase towards styrene epoxidation to date. Following this approach, a set of styrene derivatives, such as o-, m-, p-chlorostyrenes and fluorostyrenes, could also be epoxidized with modest to very good TONs (362-3480) and high (R)-enantioselectivities (95-99% ee). The semi-preparative scale synthesis of (R)-styrene oxide performed at 0 °C with high conversion, maintaining enantioselectivity, and moderate isolated yields, further suggests the potential application of the current P450 enzymatic system in styrene epoxidation. This study indicates that the synergistic use of protein engineering and an exogenous DFSM constitutes an efficient strategy to control the enantioselectivity of styrene epoxidation, thus substantially expanding the chemical scope of P450 enzymes as useful bio-oxidative catalysts.

| S-EPMC8115292 | biostudies-literature

A Perspective on Late-Stage Aromatic C-H Bond Functionalization.

Project description:Late-stage functionalization of C-H bonds (C-H LSF) can provide a straightforward approach to the efficient synthesis of functionalized complex molecules. However, C-H LSF is challenging because the C-H bond must be functionalized in the presence of various other functional groups. In this Perspective, we evaluate aromatic C-H LSF on the basis of four criteria─reactivity, chemoselectivity, site-selectivity, and substrate scope─and provide our own views on current challenges as well as promising strategies and areas of growth going forward.

| S-EPMC8855345 | biostudies-literature

An efficient light-driven P450 BM3 biocatalyst.

Project description:P450s are heme thiolate enzymes that catalyze the regio- and stereoselective functionalization of unactivated C-H bonds using molecular dioxygen and two electrons delivered by the reductase. We have developed hybrid P450 BM3 heme domains containing a covalently attached Ru(II) photosensitizer in order to circumvent the dependency on the reductase and perform P450 reactions upon visible light irradiation. A highly active hybrid enzyme with improved stability and a modified Ru(II) photosensitizer is able to catalyze the light-driven hydroxylation of lauric acid with total turnover numbers of 935 and initial reaction rate of 125 mol product/(mol enzyme/min).

| S-EPMC3938315 | 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