Obafluorin
目录号 : GC45617A β-lactone antibiotic
Cas No.:92121-68-1
Sample solution is provided at 25 µL, 10mM.
Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
Obafluorin is a β-lactone antibiotic that has been found in P. fluorescens.1 It is active against S. aureus, S. faecalis, K. pneumoniae, and P. vulgaris (MIC = 125 μg/ml for all).
|1. Pu, Y., Lowe, C., Sailer, M., et al. Synthesis, stability, and antimicrobial activity of (+)-obafluorin and related β-lactone antibiotics. J. Org. Chem. 59(13), 3642-3655 (1994).
| Cas No. | 92121-68-1 | SDF | |
| Canonical SMILES | O=C(C1=C(O)C(O)=CC=C1)N[C@H]2[C@@H](CC3=CC=C([N+]([O-])=O)C=C3)OC2=O | ||
| 分子式 | C17H14N2O7 | 分子量 | 358.3 |
| 溶解度 | Acetonitrile: >1mg/ml | 储存条件 | Store at -20°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
||
| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
 |
1 mg | 5 mg | 10 mg |
| 1 mM | 2.791 mL | 13.9548 mL | 27.9096 mL |
| 5 mM | 0.5582 mL | 2.791 mL | 5.5819 mL |
| 10 mM | 0.2791 mL | 1.3955 mL | 2.791 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Immunity-Guided Identification of Threonyl-tRNA Synthetase as the Molecular Target of Obafluorin, a β-Lactone Antibiotic
ACS Chem Biol 2019 Dec 20;14(12):2663-2671.PMID:31675206DOI:10.1021/acschembio.9b00590.
To meet the ever-growing demands of antibiotic discovery, new chemical matter and antibiotic targets are urgently needed. Many potent natural product antibiotics which were previously discarded can also provide lead molecules and drug targets. One such example is the structurally unique β-lactone Obafluorin, produced by Pseudomonas fluorescens ATCC 39502. Obafluorin is active against both Gram-positive and -negative pathogens; however, the biological target was unknown. We now report that Obafluorin targets threonyl-tRNA synthetase, and we identify a homologue, ObaO, which confers immunity to the Obafluorin producer. Disruption of obaO in P. fluorescens ATCC 39502 results in Obafluorin sensitivity, whereas expression in sensitive E. coli strains confers resistance. Enzyme assays demonstrate that E. coli threonyl-tRNA synthetase is fully inhibited by Obafluorin, whereas ObaO is only partly susceptible, exhibiting a very unusual partial inhibition mechanism. Altogether, our data highlight the utility of an immunity-guided approach for the identification of an antibiotic target de novo and will ultimately enable the generation of improved Obafluorin variants.
An L-threonine transaldolase is required for L-threo-β-hydroxy-α-amino acid assembly during Obafluorin biosynthesis
Nat Commun 2017 Jun 26;8:15935.PMID:28649989DOI:10.1038/ncomms15935.
β-Lactone natural products occur infrequently in nature but possess a variety of potent and valuable biological activities. They are commonly derived from β-hydroxy-α-amino acids, which are themselves valuable chiral building blocks for chemical synthesis and precursors to numerous important medicines. However, despite a number of excellent synthetic methods for their asymmetric synthesis, few effective enzymatic tools exist for their preparation. Here we report cloning of the biosynthetic gene cluster for the β-lactone antibiotic Obafluorin and delineate its biosynthetic pathway. We identify a nonribosomal peptide synthetase with an unusual domain architecture and an L-threonine:4-nitrophenylacetaldehyde transaldolase responsible for (2S,3R)-2-amino-3-hydroxy-4-(4-nitrophenyl)butanoate biosynthesis. Phylogenetic analysis sheds light on the evolutionary origin of this rare enzyme family and identifies further gene clusters encoding L-threonine transaldolases. We also present preliminary data suggesting that L-threonine transaldolases might be useful for the preparation of L-threo-β-hydroxy-α-amino acids.
l-Threonine Transaldolase Activity Is Enabled by a Persistent Catalytic Intermediate
ACS Chem Biol 2021 Jan 15;16(1):86-95.PMID:33337128DOI:10.1021/acschembio.0c00753.
l-Threonine transaldolases (lTTAs) are a poorly characterized class of pyridoxal-5'-phosphate (PLP) dependent enzymes responsible for the biosynthesis of diverse β-hydroxy amino acids. Here, we study the catalytic mechanism of ObiH, an lTTA essential for biosynthesis of the β-lactone natural product Obafluorin. Heterologously expressed ObiH purifies as a mixture of chemical states including a catalytically inactive form of the PLP cofactor. Photoexcitation of ObiH promotes the conversion of the inactive state of the enzyme to the active form. UV-vis spectroscopic analysis reveals that ObiH catalyzes the retro-aldol cleavage of l-threonine to form a remarkably persistent glycyl quinonoid intermediate, with a half-life of ∼3 h. Protonation of this intermediate is kinetically disfavored, enabling on-cycle reactivity with aldehydes to form β-hydroxy amino acids. We demonstrate the synthetic potential of ObiH via the single step synthesis of (2S,3R)-β-hydroxyleucine. To further understand the structural features underpinning this desirable reactivity, we determined the crystal structure of ObiH bound to PLP as the Schiff's base at 1.66 Å resolution. This high-resolution model revealed a unique active site configuration wherein the evolutionarily conserved Asp that traditionally H-bonds to the cofactor is swapped for a neighboring Glu. Molecular dynamics simulations combined with mutagenesis studies indicate that a structural rearrangement is associated with l-threonine entry into the catalytic cycle. Together, these data explain the basis for the unique reactivity of lTTA enzymes and provide a foundation for future engineering and mechanistic analysis.
The structural basis of N-acyl-α-amino-β-lactone formation catalyzed by a nonribosomal peptide synthetase
Nat Commun 2019 Jul 31;10(1):3432.PMID:31366889DOI:10.1038/s41467-019-11383-7.
Nonribosomal peptide synthetases produce diverse natural products using a multidomain architecture where the growing peptide, attached to an integrated carrier domain, is delivered to neighboring catalytic domains for bond formation and modification. Investigation of these systems can lead to the discovery of new structures, unusual biosynthetic transformations, and to the engineering of catalysts for generating new products. The antimicrobial β-lactone Obafluorin is produced nonribosomally from dihydroxybenzoic acid and a β-hydroxy amino acid that cyclizes into the β-lactone during product release. Here we report the structure of the nonribosomal peptide synthetase ObiF1, highlighting the structure of the β-lactone-producing thioesterase domain and an interaction between the C-terminal MbtH-like domain with an upstream adenylation domain. Biochemical assays examine catalytic promiscuity, provide mechanistic insight, and demonstrate utility for generating Obafluorin analogs. These results advance our understanding of the structural cycle of nonribosomal peptide synthetases and provide insights into the production of β-lactone natural products.
Tyrosine-targeted covalent inhibition of a tRNA synthetase aided by zinc ion
Commun Biol 2023 Jan 27;6(1):107.PMID:36707692DOI:10.1038/s42003-023-04517-7.
Aminoacyl-tRNA synthetases (AARSs), a family of essential protein synthesis enzymes, are attractive targets for drug development. Although several different types of AARS inhibitors have been identified, AARS covalent inhibitors have not been reported. Here we present five unusual crystal structures showing that threonyl-tRNA synthetase (ThrRS) is covalently inhibited by a natural product, Obafluorin (OB). The residue forming a covalent bond with OB is a tyrosine in ThrRS active center, which is not commonly modified by covalent inhibitors. The two hydroxyl groups on the o-diphenol moiety of OB form two coordination bonds with the conserved zinc ion in the active center of ThrRS. Therefore, the β-lactone structure of OB can undergo ester exchange reaction with the phenolic group of the adjacent tyrosine to form a covalent bond between the compound and the enzyme, and allow its nitrobenzene structure to occupy the binding site of tRNA. In addition, when this tyrosine was replaced by a lysine or even a weakly nucleophilic arginine, similar bonds could also be formed. Our report of the mechanism of a class of AARS covalent inhibitor targeting multiple amino acid residues could facilitate approaches to drug discovery for cancer and infectious diseases.