Abstract：
:The anticancer molecule taxol (Paclitaxel) stands as one of the most medically and economically important natural products. However, despite decades of extensive study, its biosynthesis remains poorly understood. Unpredictable behavior of the first oxygenation enzyme, taxadiene-5α-hydroxylase, which produces a range of undesired products, currently stands as a key bottleneck to improved taxol production. We herein present chemical and biological evidence of an unreported epoxidase activity of taxadiene-5α-hydroxylase that puts into question the previously proposed radical-rebound mechanism. We demonstrate that the poor selectivity of taxadiene-5α-hydroxylase arises from nonselective degradation of an epoxide intermediate produced via a selective oxidation step, rather than from promiscuous oxidation, as previously proposed. We support these conclusions by demonstrating variable enzyme behavior in differing hosts and conditions, similarity of products and product ratios generated from chemical epoxidation, and taxadiene-5α-hydroxylase, and differing enzymatic activity on alternative taxadiene isomers. Additionally, we use directed mutagenesis to describe the oxidizing species of the P450, demonstrate that further in vivo functionalization of oxidized taxadiene is unable to improve selectivity of the oxidation, and show that multiple products are produced in the Taxus cuspidata and are not simply an artifact of heterologous expression. Our results highlight an important, and previously unknown, obstacle to improved taxol production. We further offer insights to overcome the challenges posed by an epoxide-mediated reaction, which sets the basis for further engineering of taxol biosynthesis.
journal_name
ACS Chem Bioljournal_title
ACS chemical biologyauthors
Edgar S,Zhou K,Qiao K,King JR,Simpson JH,Stephanopoulos Gdoi
10.1021/acschembio.5b00767subject
Has Abstractpub_date
2016-02-19 00:00:00pages
460-9issue
2eissn
1554-8929issn
1554-8937journal_volume
11pub_type
杂志文章abstract：:Cell-permeable small molecules can be used to modulate protein function selectively, rapidly, reversibly, and conditionally with temporal and quantitative control in biological systems. The identification of these chemical probes can require the screening of large numbers of small molecules. With the advent of new tec...
journal_title：ACS chemical biology
pub_type： 杂志文章,评审
doi：10.1021/cb600321j
更新日期：2007-01-23 00:00:00
abstract：:Alzheimer's Disease (AD) is a progressive neurodegenerative disease and the most common cause of dementia. The current treatment options for AD are limited to ameliorating cognitive decline temporarily and not reversing or preventing the progression of dementia. Hence, more effective therapeutic strategies are needed ...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/acschembio.0c00851
更新日期：2020-12-18 00:00:00
abstract：:Recent studies have shown that cholesterol plays a significant role in the ability of Toxin A from Clostridium difficile to enter eukaryotic cells. The translocation process is one of three major steps during intoxication that could be targeted for intervention against the severe antibiotic-associated diarrhea caused ...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/cb600133b
更新日期：2006-04-25 00:00:00
abstract：:The ubiquitin proteasome system is widely postulated to be a new and important field of drug discovery for the future, with the ubiquitin specific proteases (USPs) representing one of the more attractive target classes within the area. Many USPs have been linked to critical axes for therapeutic intervention, and the f...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/acschembio.7b00334
更新日期：2017-12-15 00:00:00
abstract：:The complexity of the human proteome is greatly expanded by post-translational modifications. New tools capable of recognizing these modifications in a sequence-specific fashion provide a route to purify these modified proteins, to alter protein trafficking, and to visualize signal transduction in real time. Here, we ...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/cb800069c
更新日期：2008-08-15 00:00:00
abstract：:The mitochondrial ADP/ATP carrier (AAC) is a prominent actor in the energetic regulation of the cell, importing ADP into the mitochondria and exporting ATP toward the cytoplasm. Severe genetic diseases have been ascribed to specific mutations in this membrane protein. How minute, well-localized modifications of the tr...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/cb300012j
更新日期：2012-07-20 00:00:00
abstract：:Protein phosphatases are critical regulators of cellular signaling in both eukaryotes and prokaryotes. The majority of protein phosphatases dephosphorylate phosphoserine/phosphothreonine or phosphotyrosine residues. Recently, however, YwlE, a member of the low-molecular weight protein tyrosine phosphatase (LMW-PTP) fa...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/cb4001469
更新日期：2013-09-20 00:00:00
abstract：:Phosphatases play key roles in normal physiology and diseases. Studying phosphatases has been both essential and challenging, and the application of conventional genetic and biochemical methods has led to crucial but still limited understanding of their mechanisms, substrates, and exclusive functions within highly int...
journal_title：ACS chemical biology
pub_type： 杂志文章,评审
doi：10.1021/acschembio.6b00570
更新日期：2016-11-18 00:00:00
abstract：:Staphylococcus aureus (S. aureus) is a Gram-positive bacterial pathogen that has emerged as a major public health threat. Here we report that the cell wall of S. aureus can be covalently re-engineered to contain non-native small molecules. This process makes use of endogenous levels of the bacterial enzyme sortase A (...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/cb100195d
更新日期：2010-12-17 00:00:00
abstract：:C-methylation of aromatic small molecules by C-methyltransferases (C-MTs) is an important biological transformation that involves C-C bond formation using S-adenosyl-l-methionine (SAM) as the methyl donor. Here, two advances in the mechanistic understanding of C-methylation of the 8-position of coumarin substrates cat...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/acschembio.6b01053
更新日期：2017-02-17 00:00:00
abstract：:miRNAs are small non-coding RNAs that regulate about 60% of mammalian genes by modulating their transcript levels. Network scale studies of miRNA-mediated regulatory circuits demonstrate the central importance of this class of small RNA in the maintenance of biological robustness. More recently, several reports have d...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/cb300650y
更新日期：2013-05-17 00:00:00
abstract：:SGK3 is a PX domain containing protein kinase activated at endosomes downstream of class 1 and 3 PI3K family members by growth factors and oncogenic mutations. SGK3 plays a key role in mediating resistance of breast cancer cells to class 1 PI3K or Akt inhibitors, by substituting for the loss of Akt activity and restor...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/acschembio.9b00505
更新日期：2019-09-20 00:00:00
abstract：:Bacterial tRNA-guanine transglycosylase (Tgt) is involved in the biosynthesis of the modified tRNA nucleoside queuosine present in the anticodon wobble position of tRNAs specific for aspartate, asparagine, histidine, and tyrosine. Inactivation of the tgt gene leads to decreased pathogenicity of Shigella bacteria. Ther...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/acschembio.0c00700
更新日期：2020-11-20 00:00:00
abstract：:Numerous cytochrome P450 (CYP) 2B6 substrates including drugs and environmental chemicals are halogenated. To assess the role of halogen-π bonds in substrate selectivity and orientation in the active site, structures of four CYP2B6 monoterpenoid complexes were solved by X-ray crystallography. Bornyl bromide exhibited ...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/acschembio.7b00056
更新日期：2017-05-19 00:00:00
abstract：:The hypoxia inducible factor (HIF) system is central to the signaling of low oxygen (hypoxia) in animals. The levels of HIF-α isoforms are regulated in an oxygen-dependent manner by the activity of the HIF prolyl-hydroxylases (PHD or EGLN enzymes), which are Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases. Here, ...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/cb400088q
更新日期：2013-07-19 00:00:00
abstract：:The perturbation of protein kinases with small organic molecules is a powerful approach to dissect kinase function in complex biological systems. Covalent kinase inhibitors that target thiols in the ATP binding pocket of the kinase domain proved to be ideal reagents for the investigation of highly dynamic cellular pro...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/cb200465c
更新日期：2012-04-20 00:00:00
abstract：:RNAs are involved in interaction networks with other biomolecules and are crucial for proper cell function. Yet their biochemical analysis remains challenging. For Förster Resonance Energy Transfer (FRET), a common tool to study such interaction networks, two interacting molecules have to be fluorescently labeled. "Sp...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/acschembio.7b00332
更新日期：2017-12-15 00:00:00
abstract：:To discover chemical probes to further under-stand the function of individual DNA polymerases, we established a generally applicable high-throughput screening. By applying this technique we discovered three novel inhibitor classes of human DNA polymerase λ (DNA Pol λ), a key enzyme to maintain the genetic integrity of...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/cb100382m
更新日期：2011-04-15 00:00:00
abstract：:The inhibitor of apoptosis (IAP) proteins are critical regulators of cancer cell survival, which makes them attractive targets for therapeutic intervention in cancers. Herein, we describe the structure-based design of IAP antagonists with high affinities and selectivity (>2000-fold) for c-IAP1 over XIAP and their func...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/cb900083m
更新日期：2009-07-17 00:00:00
abstract：:Histo-blood group epitopes are fucosylated branched oligosaccharides with well-defined conformations in solution that are recognized by receptors, such as lectins from pathogens. We report here the results of a series of experimental and computational endeavors revealing the unusual distortion of histo-blood group ant...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/acschembio.6b00333
更新日期：2016-07-15 00:00:00
abstract：:Light-activated protein domains provide a convenient, modular, and genetically encodable sensor for optogenetics and optobiology. Although these domains have now been deployed in numerous systems, the precise mechanism of photoactivation and the accompanying structural dynamics that modulate output domain activity rem...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/acschembio.0c00543
更新日期：2020-10-16 00:00:00
abstract：:In eukaryotic cells, proteins and RNAs are transported between the nucleus and the cytoplasm by nuclear import and export receptors. Over the past decade, small molecules that inhibit the nuclear export receptor CRM1 have been identified, most notably leptomycin B. However, up to now no small molecule inhibitors of nu...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/cb100094k
更新日期：2010-10-15 00:00:00
abstract：:Regulators of G protein signaling (RGS) proteins are key players in regulating signaling via G protein-coupled receptors. RGS proteins directly bind to the Gα-subunits of activated heterotrimeric G-proteins, and accelerate the rate of GTP hydrolysis, thereby rapidly deactivating G-proteins. Using atomistic simulations...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/cb400568g
更新日期：2013-12-20 00:00:00
abstract：:Peptidoglycan glycosyltransferases (PGTs), enzymes that catalyze the formation of the glycan chains of the bacterial cell wall, have tremendous potential as antibiotic targets. The moenomycins, a potent family of natural product antibiotics, are the only known active site inhibitors of the PGTs and serve as blueprints...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/cb800078a
更新日期：2008-07-18 00:00:00
abstract：:Drug optimization is guided by biophysical methods with increasing popularity. In the context of lead structure modifications, the introduction of methyl groups is a simple but potentially powerful approach. Hence, it is crucial to systematically investigate the influence of ligand methylation on biophysical character...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/acschembio.9b00476
更新日期：2019-12-20 00:00:00
abstract：:In a process called quorum sensing, bacteria produce and secrete certain signaling compounds (called autoinducers) that bind to receptors on other bacteria and activate transcription of certain genes. A clever genetic selection yields a new quorum-sensing transcriptional regulator that marches to the beat of a differe...
journal_title：ACS chemical biology
pub_type： 杂志文章,评审
doi：10.1021/cb6003417
更新日期：2006-08-22 00:00:00
abstract：:Chloropupukeananin and chloropestolides are novel metabolites of the plant endophyte Pestalotiopsis fici, showing antimicrobial, antitumor, and anti-HIV activities. Their highly complex and unique skeletons were generated from the coisolated pestheic acid (1) and iso-A82775C (10) based on our previous studies. Here, w...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/acschembio.7b01059
更新日期：2018-03-16 00:00:00
abstract：:Conotoxins are small disulfide-rich peptides from the venom of cone snails. Along with other conopeptides, they target a wide range of membrane receptors, ion channels, and transporters, and because of their high potency and selectivity for defined subtypes of these receptors, they have attracted a great deal of atten...
journal_title：ACS chemical biology
pub_type： 杂志文章,评审
doi：10.1021/cb700091j
更新日期：2007-07-20 00:00:00
abstract：:Lasso peptides are bacterial ribosomally synthesized and post-translationally modified peptides. They have sparked increasing interest in peptide-based drug development because of their compact, interlocked structure, which offers superior stability and protein-binding capacity. Disulfide bond-containing lasso peptide...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/acschembio.5b00584
更新日期：2015-11-20 00:00:00
abstract：:A new chemical method for the traceless labeling of glycoproteins with synthetic boronic acid (BA)-tosyl probes was successfully developed. The BA moiety acts as an affinity head to direct the formation of a cyclic boronate diester with the diol groups of glycans. Following this step, the electrophilic tosyl group is ...
journal_title：ACS chemical biology
pub_type： 杂志文章
doi：10.1021/cb400631w
更新日期：2014-02-21 00:00:00
