The Kalimantacin Polyketide Antibiotics Inhibit Fatty Acid Biosynthesis in Staphylococcus aureus by Targeting the Enoyl‐Acyl Carrier Protein Binding Site of FabI

The enoyl‐acyl carrier protein reductase enzyme FabI is essential for fatty acid biosynthesis in Staphylococcus aureus and represents a promising target for the development of novel, urgently needed anti‐staphylococcal agents. Here, we elucidate the mode of action of the kalimantacin antibiotics, a novel class of FabI inhibitors with clinically‐relevant activity against multidrug‐resistant S. aureus. By combining X‐ray crystallography with molecular dynamics simulations, in vitro kinetic studies and chemical derivatization experiments, we characterize the interaction between the antibiotics and their target, and we demonstrate that the kalimantacins bind in a unique conformation that differs significantly from the binding mode of other known FabI inhibitors. We also investigate mechanisms of acquired resistance in S. aureus and identify key residues in FabI that stabilize the binding of the antibiotics. Our findings provide intriguing insights into the mode of action of a novel class of FabI inhibitors that will inspire future anti‐staphylococcal drug development.     

The Kalimantacin Polyketide Antibiotics Inhibit Fatty Acid Biosynthesis in Staphylococcus aureus by Targeting the Enoyl-Acyl Carrier Protein Binding Site of FabI

The enoyl-acyl carrier protein reductase enzyme FabI is essential for fatty acid biosynthesis in Staphylococcus aureus and represents a promising target for the development of novel, urgently needed anti-staphylococcal agents. Here, we elucidate the mode of action of the kalimantacin antibiotics, a novel class of FabI inhibitors with clinically-relevant activity against multidrug-resistant S. aureus. By combining X-ray crystallography with molecular dynamics simulations, in vitro kinetic studies and chemical derivatization experiments, we characterize the interaction between the antibiotics and their target, and we demonstrate that the kalimantacins bind in a unique conformation that differs significantly from the binding mode of other known FabI inhibitors. We also investigate mechanisms of acquired resistance in S. aureus and identify key residues in FabI that stabilize the binding of the antibiotics. Our findings provide intriguing insights into the mode of action of a novel class of FabI inhibitors that will inspire future anti-staphylococcal drug development.     

The Molecular Basis of Catalysis by SDR Family Members Ketoacyl-ACP Reductase FabG and Enoyl-ACP Reductase FabI in Type-II Fatty Acid Biosynthesis

The short-chain dehydrogenase/reductase (SDR) superfamily members acyl-ACP reductases FabG and FabI are indispensable core enzymatic modules and catalytic orientation controllers in type-II fatty acid biosynthesis. Herein, we report their distinct substrate allosteric recognition and enantioselective reduction mechanisms. FabG achieves allosteric regulation of ACP and NADPH through ACP binding across two adjacent FabG monomers, while FabI follows an irreversible compulsory order of substrate binding in that NADH binding must precede that of ACP on a discrete FabI monomer. Moreover, FabG and FabI utilize a backdoor residue Phe187 or a “rheostat” α8 helix for acyl chain length selection, and their corresponding triad residues Ser142 or Tyr145 recognize the keto- or enoyl-acyl substrates, respectively, facilitating initiation of nucleophilic attack by NAD(P)H. The other two triad residues (Tyr and Lys) mediate subsequent proton transfer and (R)-3-hydroxyacyl- or saturated acyl-ACP production.     

Control of β‐Branching in Kalimantacin Biosynthesis: Application of 13C NMR to Polyketide Programming

The presence of β‐branches in the structure of polyketides that possess potent biological activity underpins the widespread importance of this structural feature. Kalimantacin is a polyketide antibiotic with selective activity against staphylococci, and its biosynthesis involves the unprecedented incorporation of three different and sequential β‐branching modifications. We use purified single and multi‐domain enzyme components of the kalimantacin biosynthetic machinery to address in vitro how the pattern of β‐branching in kalimantacin is controlled. Robust discrimination of enzyme products required the development of a generalisable assay that takes advantage of ¹³C NMR of a single ¹³C label incorporated into key biosynthetic mimics combined with favourable dynamic properties of an acyl carrier protein. We report a previously unassigned modular enoyl‐CoA hydratase (mECH) domain and the assembly of enzyme constructs and cascades that are able to generate each specific β‐branch.     

Templated in-situ synthesis of gold nanoclusters conjugated to drug target bacterial enoyl-ACP reductase,and their application to the detection of mercury ions using a test stripe

Fluorescent gold nanoclusters (AuNCs) were synthesized using a drug target bacterial enoyl-ACP reductase (FabI) as a template. The physical and chemical properties of the AuNCs were studied by UV-vis absorption, fluorescence, X-ray photoelectron spectroscopy and TEM. The AuNCs-FabI conjugate was prepared by in situ reduction of tetrachloroaurate in the presence of FabI. The conjugated particles were loaded onto nylon membranes by taking advantage of the electrostatic interaction between the negatively charged AuNCs@FabI and the nylon film which is positively charged at pH 7.4. This results in the formation of a test stripe with sensor spots that can be used to detect Hg(II) ion in the 1 nM to 10 μM concentration range. The test stripes are simple, convenient, selective, sensitive, and can be quickly read out with bare eyes after illumination with a UV lamp.

计算机模拟三氯生对FabI活性口袋及Loop区的调控机制

应用分子动力学模拟、蛋白质二级结构定义(DSSP)和口袋体积测量(POVME)计算方法分别研究了FabI(烯脂酰-ACP还原酶)-NAD+(氧化型烟酰胺腺嘌呤二核苷酸)二元复合物和FabI-NAD+-TCL(三氯生)三元复合物体系中活性口袋loop区构象、loop区二级结构、活性口袋体积以及底物(酰基不饱和链)通道随模拟时间的变化规律.研究表明,在FabI-NAD+-TCL三元复合物中,三氯生限制了活性口袋及底物通道的变化,活性口袋loop区呈现为规则、闭合的稳定构象,位于活性口袋正前方,常伴有螺旋二级结构的形成,从而使活性口袋体积变化较小、分布比较集中,底物通道较窄或处于关闭状态.而在FabI-NAD+二元复合物中,活性口袋loop区以无规则、开启的柔性构象存在,活性口袋体积变化较大,分布比较分散,底物通道明显较宽且不稳定.可见,三氯生能诱导FabI活性口袋及loop区的构象变化,使活性口袋构成了紧密的统一体、封闭了底物通道,从而阻碍了酰基不饱和链通过底物通道进入蛋白酶的催化中心,中断了该酶催化的还原反应和细菌脂肪酸合成循环.上述发现对深入认识三氯生的抗菌作用机制及相关药物的改良与设计具有重要的指导意义.     

计算机模拟三氯生对FabI活性口袋及Loop区的调控机制

应用分子动力学模拟、蛋白质二级结构定义(DSSP)和口袋体积测量(POVME)计算方法分别研究了FabI (烯脂酰-ACP还原酶)-NAD⁺(氧化型烟酰胺腺嘌呤二核苷酸)二元复合物和FabI-NAD⁺-TCL(三氯生)三元复合物体系中活性口袋loop 区构象、loop 区二级结构、活性口袋体积以及底物(酰基不饱和链)通道随模拟时间的变化规律. 研究表明,在FabI-NAD⁺-TCL三元复合物中,三氯生限制了活性口袋及底物通道的变化,活性口袋loop 区呈现为规则、闭合的稳定构象,位于活性口袋正前方,常伴有螺旋二级结构的形成,从而使活性口袋体积变化较小、分布比较集中,底物通道较窄或处于关闭状态. 而在FabI-NAD⁺二元复合物中,活性口袋loop 区以无规则、开启的柔性构象存在,活性口袋体积变化较大,分布比较分散,底物通道明显较宽且不稳定. 可见,三氯生能诱导FabI 活性口袋及loop 区的构象变化,使活性口袋构成了紧密的统一体、封闭了底物通道,从而阻碍了酰基不饱和链通过底物通道进入蛋白酶的催化中心,中断了该酶催化的还原反应和细菌脂肪酸合成循环. 上述发现对深入认识三氯生的抗菌作用机制及相关药物的改良与设计具有重要的指导意义.     

Evaluating thermodynamic integration performance of the new amber molecular dynamics package and assess potential halogen bonds of enoyl‐ACP reductase (FabI) benzimidazole inhibitors

Thermodynamic integration (TI) can provide accurate binding free energy insights in a lead optimization program, but its high computational expense has limited its usage. In the effort of developing an efficient and accurate TI protocol for FabI inhibitors lead optimization program, we carefully compared TI with different Amber molecular dynamics (MD) engines (sander and pmemd), MD simulation lengths, the number of intermediate states and transformation steps, and the Lennard‐Jones and Coulomb Softcore potentials parameters in the one‐step TI, using eleven benzimidazole inhibitors in complex with Francisella tularensis enoyl acyl reductase (FtFabI). To our knowledge, this is the first study to extensively test the new AMBER MD engine, pmemd, on TI and compare the parameters of the Softcore potentials in the one‐step TI in a protein‐ligand binding system. The best performing model, the one‐step pmemd TI, using 6 intermediate states and 1 ns MD simulations, provides better agreement with experimental results (RMSD = 0.52 kcal/mol) than the best performing implicit solvent method, QM/MM‐GBSA from our previous study (RMSD = 3.00 kcal/mol), while maintaining similar efficiency. Briefly, we show the optimized TI protocol to be highly accurate and affordable for the FtFabI system. This approach can be implemented in a larger scale benzimidazole scaffold lead optimization against FtFabI. Lastly, the TI results here also provide structure‐activity relationship insights, and suggest the parahalogen in benzimidazole compounds might form a weak halogen bond with FabI, which is a well‐known halogen bond favoring enzyme. © 2015 Wiley Periodicals, Inc.     

Recent advances in antimicrobial nucleoside antibiotics targeting cell wall biosynthesis

The biosynthesis of peptidoglycan is essential for all bacteria and has no counterpart in eukaryotic cells. It is one of the prime targets for antibiotic chemotherapy, especially phospho-MurNAc-pentapeptide translocase (translocase I) is a fascinating target in which there is no commercial antibiotic. In this review we will describe three nucleoside translocase I inhibitors, mureidomycin, tunicamycin and liposidomycin.     

硒蛋白的分子生物学及与疾病的关系*

硒蛋白是微量元素硒在体内存在和发挥生物功能的主要形式。因硒蛋白的活性中心硒代半胱氨酸由传统终止码TGA编码,故从基因组中预测硒蛋白以及用基因工程技术表达硒蛋白均很困难。有关硒抗氧化、对癌症、神经退行性疾病和病毒作用的报导较多,但结论并不一致。本文综述了硒蛋白基因预测、蛋白质表达调控以及硒和硒蛋白对癌症、神经退行性疾病和病毒的作用及机制等方面的近期进展,研究提高硒蛋白生物信息学预测准确率和基因工程表达量的方法,分析了解硒蛋白与疾病发生发展的关系和机制,探索不同硒蛋白作为预防药物开发、作为癌症治疗和药物筛选靶标的可能性。     

Comparison of radii sets,entropy, QM methods,and sampling on MM‐PBSA,MM‐GBSA,and QM/MM‐GBSA ligand binding energies of F. tularensis enoyl‐ACP reductase (FabI)

To validate a method for predicting the binding affinities of FabI inhibitors, three implicit solvent methods, MM‐PBSA, MM‐GBSA, and QM/MM‐GBSA were carefully compared using 16 benzimidazole inhibitors in complex with Francisella tularensis FabI. The data suggests that the prediction results are sensitive to radii sets, GB methods, QM Hamiltonians, sampling protocols, and simulation length, if only one simulation trajectory is used for each ligand. In this case, QM/MM‐GBSA using 6 ns MD simulation trajectories together with GB^neck2, PM3, and the mbondi2 radii set, generate the closest agreement with experimental values (r² = 0.88). However, if the three implicit solvent methods are averaged from six 1 ns MD simulations for each ligand (called “multiple independent sampling”), the prediction results are relatively insensitive to all the tested parameters. Moreover, MM/GBSA together with GB^HCT and mbondi, using 600 frames extracted evenly from six 0.25 ns MD simulations, can also provide accurate prediction to experimental values (r² = 0.84). Therefore, the multiple independent sampling method can be more efficient than a single, long simulation method. Since future scaffold expansions may significantly change the benzimidazole's physiochemical properties (charges, etc.) and possibly binding modes, which may affect the sensitivities of various parameters, the relatively insensitive “multiple independent sampling method” may avoid the need of an entirely new validation study. Moreover, due to large fluctuating entropy values, (QM/)MM‐P(G)BSA were limited to inhibitors’ relative affinity prediction, but not the absolute affinity. The developed protocol will support an ongoing benzimidazole lead optimization program. © 2015 Wiley Periodicals, Inc.     

YBR246W is required for the third step of diphthamide biosynthesis

Diphthamide, the target of diphtheria toxin, is a post-translationally modified histidine residue that is found in archaeal and eukaryotic translation elongation factor 2. The biosynthesis and function of this modification has attracted the interest of many biochemists for decades. The biosynthesis has been known to proceed in three steps. Proteins required for the first and second steps have been identified, but the protein(s) required for the last step have remained elusive. Here we demonstrate that the YBR246W gene in yeast is required for the last step of diphthamide biosynthesis, as the deletion of YBR246W leads to the accumulation of diphthine, which is the enzymatic product of the second step of the biosynthesis. This discovery will provide important information leading to the complete elucidation of the full biosynthesis pathway of diphthamide.     

Why does β-secretase zymogen possess catalytic activity? Molecular modeling and molecular dynamics simulation studies

Beta-secretase is a potential target for inhibitory drugs against Alzheimer's disease as it cleaves amyloid precursor protein (APP) to form insoluble amyloid plaques and vascular deposits in the brain. Beta-secretase is matured from its precursor protein, called beta-secretase zymogen, which, different from most of other zymogens, is also partially active in cleaving APP. Hence, it is important to study on the mechanism of the zymogen's activation process. This study was to model the 3-D structure of the zymogen, followed by intensive molecular dynamics (MD) simulations to identify the most probable 3-D model and to study the dynamic structural behavior of the zymogen for understanding the effects of pro-segment on the function of the enzyme. The results revealed that the dropping in catalytic activity of the beta-secretase zymogen could be attributed to the occupation of the entrance of the catalytic site of the zymogen by its pro-segment. On the other hand, the partial catalytic activity of the zymogen could be explained by high fluctuation of the pro-segment in comparison with that of other zymogens, resulting in the occasionally exposure of the catalytic site for access its substrate APP. Indeed, steered MD (SMD) simulation revealed a weak pulling force at quasi-equilibrium state for the pro-segment of the zymogen leaving from the entrance, indicating that this swinging process could take place spontaneously. Furthermore, MM-PBSA calculation revealed a small change of free energy of 10.56 kal/mol between the initial and final states of the process of pro-segment swung outside the binding pocket of beta-secretase zymogen. These results not only account for the partial catalytic activity of beta-secretase zymogen, but also provide useful clues for discovering new potent ligands, as new type of drug leads for curing Alzheimer's disease, to prevent the pro-segment of the zymogen from leaving its catalytic site.     

Δ(11,12) double bond formation in tirandamycin biosynthesis is atypically catalyzed by TrdE, a glycoside hydrolase family enzyme

The tirandamycins (TAMs) are a small group of Streptomyces-derived natural products that target bacterial RNA polymerase. Within the TAM biosynthetic cluster, trdE encodes a glycoside hydrolase whose role in TAM biosynthesis has been undefined until now. We report that in vivo trdE inactivation leads to accumulation of pre-tirandamycin, the earliest intermediate released from its mixed polyketide/nonribosomal peptide biosynthetic assembly line. In vitro and site-directed mutagenesis studies showed that TrdE, a putative glycoside hydrolase, catalyzes in a highly atypical fashion the installation of the Δ(11,12) double bond during TAM biosynthesis.     

Exploring modular reengineering strategies to redesign the teicoplanin non-ribosomal peptide synthetase

Non-ribosomal peptide synthesis is an important biosynthesis pathway in secondary metabolism. In this study we have investigated modularisation and redesign strategies for the glycopeptide antibiotic teicoplanin. Using the relocation or exchange of domains within the NRPS modules, we have identified how to initiate peptide biosynthesis and explored the requirements for the functional reengineering of both the condensation/adenylation domain and epimerisation/condensation domain interfaces. We have also demonstrated strategies that ensure communication between isolated NRPS modules, leading to new peptide assembly pathways. This provides important insights into NRPS reengineering of glycopeptide antibiotic biosynthesis and has broad implications for the redesign of other NRPS systems.

Redesign of the non-ribosomal peptide synthetase (NRPS) from teicoplanin biosynthesis has been extensively investigated via domain exchange, interface reengineering and through engineering communication between isolated NRPS modules.     

Total Synthesis of Dansylated Park's Nucleotide for High‐Throughput MraY Assays

The membrane protein translocase I (MraY) is a key enzyme in bacterial peptidoglycan biosynthesis. It is therefore frequently discussed as a target for the development of novel antibiotics. The screening of compound libraries for the identification of MraY inhibitors is enabled by an established fluorescence‐based MraY assay. However, this assay requires a dansylated derivative of the bacterial biosynthetic intermediate Park's nucleotide as the MraY substrate. Isolation of Park's nucleotide from bacteria and subsequent dansylation only furnishes limited amounts of this substrate, thus hampering the high‐throughput screening for MraY inhibitors. Accordingly, the efficient provision of dansylated Park's nucleotide is a major bottleneck in the exploration of this promising drug target. In this work, we present the first total synthesis of dansylated Park's nucleotide, affording an unprecedented amount of the target compound for high‐throughput MraY assays.     

Toward the pathway of S. aureus WTA biosynthesis

Wall teichoic acid (WTA) contributes profoundly to the virulence of Staphylococcus aureus. The successful in vitro reconstitution of poly-ribitolphosphate WTA biosynthesis using recombinant enzymes sheds new light on WTA enzymology and paves the way for developing new antibiotics that target WTA biosynthesis, as discussed in Brown et al. in a recent issue of Chemistry & Biology.     

A streamlined metabolic pathway for the biosynthesis of moenomycin A

Moenomycin A (MmA) is a member of the phosphoglycolipid family of antibiotics, which are the only natural products known to directly target the extracellular peptidoglycan glycosyltransferases involved in bacterial cell wall biosynthesis. The structural and biological uniqueness of MmA make it an attractive starting point for the development of new antibacterial drugs. In order both to elucidate the biosynthesis of this unusual compound and to develop tools to manipulate its structure, we have identified the MmA biosynthetic genes in Streptomyces ghanaensis (ATCC14672). We show via heterologous expression of a subset of moe genes that the economy of the MmA pathway is enabled through the use of sugar-nucleotide and isoprenoid building blocks derived from primary metabolism. The work reported lays the foundation for genetic engineering of MmA biosynthesis to produce novel derivatives.     

The biosynthetic gene cluster for the antitumor rebeccamycin: characterization and generation of indolocarbazole derivatives

Rebeccamycin, a halogenated natural product of the indolocarbazole family, is produced by Saccharothrix aerocolonigenes ATCC39243. Several rebeccamycin analogues, which target DNA topoisomerase I or II, have already entered clinical trials as anticancer drugs. Using as a probe an internal fragment of ngt, a Saccharothrix aerocolonigenes gene encoding an indolocarbazole N-glycosyltransferase, we isolated a DNA region that directed the biosynthesis of rebeccamycin when introduced into Streptomyces albus. Sequence analysis of 25.6 kb revealed genes for indolocarbazole core formation, halogenation, glycosylation, and sugar methylation, as well as a regulatory gene and two resistance/secretion genes. Heterologous expression of subsets of these genes resulted in production of deschloro-rebeccamycin, 4'-demethyldeschloro-rebeccamycin, and deschloro-rebeccamycin aglycone. The cloned genes should help to elucidate the molecular basis for indolocarbazole biosynthesis and set the stage for the generation of novel indolocarbazole analogues by genetic engineering.     

Biosynthesis of the Apoptolidins in Nocardiopsis sp. FU 40

The apoptolidins are 20/21-membered macrolides produced by Nocardiopsis sp. FU40. Several members of this family are potent and remarkably selective inducers of apoptosis in cancer cell lines, likely via a distinct mitochondria associated target. To investigate the biosynthesis of this natural product, the complete genome of the apoptolidin producer Nocardiopsis sp. FU40 was sequenced and a 116 kb region was identified containing a putative apoptolidin biosynthetic gene cluster. The apoptolidin gene cluster comprises a type I polyketide synthase, with 13 homologating modules, apparently initiated in an unprecedented fashion via transfer from a methoxymalonyl-acyl carrier protein loading module. Spanning approximately 39 open reading frames, the gene cluster was cloned into a series of overlapping cosmids and functionally validated by targeted gene disruption experiments in the producing organism. Disruption of putative PKS and P₄₅₀ genes delineated the roles of these genes in apoptolidin biosynthesis and chemical complementation studies demonstrated intact biosynthesis peripheral to the disrupted genes. This work provides insight into details of the biosynthesis of this biologically significant natural product and provides a basis for future mutasynthetic methods for the generation of non-natural apoptolidins.