Assessing Boron-Pleuromutilin AN11251 for the Development of Antibacterial Agents.

Pleuromutilins are a group of antibiotics derived from the naturally occurring compound. The recent approval of lefamulin for both intravenous and oral doses in humans to treat community-acquired bacterial pneumonia has prompted investigations in modifying the structure to broaden the antibacterial spectrum, enhance the activity, and improve the pharmacokinetic properties. AN11251 is a C(14)-functionalized pleuromutilin with a boron-containing heterocycle substructure. It was demonstrated to be an anti-Wolbachia agent with therapeutic potential for Onchocerciasis and lymphatic filariasis. Here, the in vitro and in vivo PK parameters of AN11251 were measured including PPB, intrinsic clearance, half-life, systemic clearance, and volume of distribution. The results indicate that the benzoxaborole-modified pleuromutilin possesses good ADME and PK properties. AN11251 has potent activities against the Gram-positive bacterial pathogens tested, including various drug-resistant strains, and against the slow-growing mycobacterial species. Finally, we employed PK/PD modeling to predict the human dose for treatment of disease caused by Wolbachia, Gram-positive bacteria, or Mycobacterium tuberculosis, which might facilitate the further development of AN11251.

Re-discovery of PF-3845 as a new chemical scaffold inhibiting phenylalanyl-tRNA synthetase in Mycobacterium tuberculosis.

Mycobacteria tuberculosis (Mtb) remains the deadliest pathogenic bacteria worldwide. The search for new antibiotics to treat drug-sensitive as well as drug-resistant tuberculosis has become a priority. The essential enzyme phenylalanyl-tRNA synthetase (PheRS) is an antibacterial drug target because of the large differences between bacterial and human PheRS counterparts. In a high-throughput screening of 2148 bioactive compounds, PF-3845, which is a known inhibitor of human fatty acid amide hydrolase (FAAH), was identified inhibiting Mtb PheRS at Ki ~0.73 ± 0.06 µM. The inhibition mechanism was studied with enzyme kinetics, protein structural modelling and crystallography, in comparison to a PheRS inhibitor of the noted phenyl-thiazolylurea-sulfonamide class. The 2.3-Å crystal structure of Mtb PheRS in complex with PF-3845 revealed its novel binding mode, in which a trifluoromethyl-pyridinylphenyl group occupies the Phe pocket while a piperidine-piperazine urea group binds into the ATP pocket through an interaction network enforced by a sulfate ion. It represents the first non-nucleoside bi-substrate competitive inhibitor of bacterial PheRS. PF-3845 inhibits the in vitro growth of Mtb H37Rv at ~24 µM, and the potency of PF-3845 increased against Mtb pheS-FDAS, suggesting on target activity in mycobacterial whole cells.  PF-3845 does not inhibit human cytoplasmic or mitochondrial PheRS in biochemical assay, which can be explained from the crystal structures. Further medicinal chemistry efforts focused on the piperidine-piperazine urea moiety may result in the identification of a selective antibacterial lead compound.

Rediscovery of PF-3845 as a new chemical scaffold inhibiting phenylalanyl-tRNA synthetase in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) remains the deadliest pathogenic bacteria worldwide. The search for new antibiotics to treat drug-sensitive as well as drug-resistant tuberculosis has become a priority. The essential enzyme phenylalanyl-tRNA synthetase (PheRS) is an antibacterial drug target because of the large differences between bacterial and human PheRS counterparts. In a high-throughput screening of 2148 bioactive compounds, PF-3845, which is a known inhibitor of human fatty acid amide hydrolase, was identified inhibiting Mtb PheRS at Ki ∼ 0.73 ± 0.06 µM. The inhibition mechanism was studied with enzyme kinetics, protein structural modeling, and crystallography, in comparison to a PheRS inhibitor of the noted phenyl-thiazolylurea-sulfonamide class. The 2.3-Å crystal structure of Mtb PheRS in complex with PF-3845 revealed its novel binding mode, in which a trifluoromethyl-pyridinylphenyl group occupies the phenylalanine pocket, whereas a piperidine-piperazine urea group binds into the ATP pocket through an interaction network enforced by a sulfate ion. It represents the first non-nucleoside bisubstrate competitive inhibitor of bacterial PheRS. PF-3845 inhibits the in vitro growth of Mtb H37Rv at ∼24 µM, and the potency of PF-3845 increased against an engineered strain Mtb pheS-FDAS, suggesting on target activity in mycobacterial whole cells. PF-3845 does not inhibit human cytoplasmic or mitochondrial PheRS in biochemical assay, which can be explained from the crystal structures. Further medicinal chemistry efforts focused on the piperidine-piperazine urea moiety may result in the identification of a selective antibacterial lead compound.

Controlling toughness of polymer-grafted nanoparticle composites for impact mitigation.

Toughness in an entangled polymer network is typically controlled by the number of load-bearing topological constraints per unit volume. In this work, we demonstrate a new paradigm for controlling toughness at high deformation rates in a polymer-grafted nanoparticle composite system where the entanglement density increases with the molecular mass of the graft. An unexpected peak in the toughness is observed right before the system reaches full entanglement that cannot be described through the entanglement concept alone. Quasi-elastic neutron scattering reveals enhanced segmental fluctuations of the grafts on the picosecond time scale, which propagate out to nanoparticle fluctuations on the time scale 100s of seconds as evidenced by X-ray photon correlation spectroscopy. This surprising multi-scale dissipation process suggests a nanoparticle jamming-unjamming transition. The realization that segmental dynamics can be coupled with the entanglement concept for enhanced toughness at high rates of deformation is a novel insight with relevance to the design of composite materials.

Using microprojectiles to study the ballistic limit of polymer thin films.

The dynamic impact between a particle and a planar material is important in many high impact events, and there is a growing need to characterize the mechanical properties of light-weight polymeric materials at dynamic loading conditions. Here, a laser-induced projectile impact test (LIPIT) is employed to investigate the ballistic limit (V0) and materials properties at impact velocities ranging from 40 m s-1 to 70 m s-1. An analytical expression describing the various energy dissipation mechanisms is established to estimate the yield stress and elasticity for polycarbonate thin films. This measurement approach demonstrates the utility of using low sample mass for discovery of materials for impact mitigation, as well as high-throughput mechanical characterization at dynamic loading rates.

Comparative analysis of FKBP family protein: evaluation, structure, and function in mammals and Drosophila melanogaster.

BACKGROUND: FK506-binding proteins (FKBPs) have become the subject of considerable interest in several fields, leading to the identification of several cellular and molecular pathways in which FKBPs impact prenatal development and pathogenesis of many human diseases. MAIN BODY: This analysis revealed differences between how mammalian and Drosophila FKBPs mechanisms function in relation to the immunosuppressant drugs, FK506 and rapamycin. Differences that could be used to design insect-specific pesticides. (1) Molecular phylogenetic analysis of FKBP family proteins revealed that the eight known Drosophila FKBPs share homology with the human FKBP12. This indicates a close evolutionary relationship, and possible origination from a common ancestor. (2) The known FKBPs contain FK domains, that is, a prolyl cis/trans isomerase (PPIase) domain that mediates immune suppression through inhibition of calcineurin. The dFKBP59, CG4735/Shutdown, CG1847, and CG5482 have a Tetratricopeptide receptor domain at the C-terminus, which regulates transcription and protein transportation. (3) FKBP51 and FKBP52 (dFKBP59), along with Cyclophilin 40 and protein phosphatase 5, function as Hsp90 immunophilin co-chaperones within steroid receptor-Hsp90 heterocomplexes. These immunophilins are potential drug targets in pathways associated with normal physiology and may be used to treat a variety of steroid-based diseases by targeting exocytic/endocytic cycling and vesicular trafficking. (4) By associating with presinilin, a critical component of the Notch signaling pathway, FKBP14 is a downstream effector of Notch activation at the membrane. Meanwhile, Shutdown associates with transposons in the PIWI-interacting RNA pathway, playing a crucial role in both germ cells and ovarian somas. Mutations in or silencing of dFKBPs lead to early embryonic lethality in Drosophila. Therefore, further understanding the mechanisms of FK506 and rapamycin binding to immunophilin FKBPs in endocrine, cardiovascular, and neurological function in both mammals and Drosophila would provide prospects in generating unique, insect specific therapeutics targeting the above cellular signaling pathways. CONCLUSION: This review will evaluate the functional roles of FKBP family proteins, and systematically summarize the similarities and differences between FKBP proteins in Drosophila and Mammals. Specific therapeutics targeting cellular signaling pathways will also be discussed.

pH-Controlled Electrochemical Deposition of Polyelectrolyte Complex Films.

Polyelectrolyte complex (PEC) films made from oppositely charged polymer chains have applications as drug-delivery vehicles, separation membranes, and biocompatible coatings. Conventional layer-by-layer techniques for polyelectrolyte coatings are low-throughput and multistep processes that are quite slow for building films on the order of micrometers. In this work, PEC films are electrochemically deposited using a rapid one-pot method, yielding thick (1 µm) films within short experimental time scales (5 min). This rapid electrodeposition is achieved by exploiting the reduction of hydrogen peroxide at mild electrode potentials that avoid water electrolysis yet trigger the pH-responsive self-assembly of a PEC film composed of poly(acrylic) acid and poly(allylamine) HCl. In situ rheology using an electrochemical quartz crystal microbalance quantified the shear modulus-density product of the deposited layer to be on the order of 107 Pa g/cm3 at a frequency of 15 MHz, with a viscoelastic phase angle of approximately 50°. This electrodeposition scheme furthers the development of PEC coatings for more high-throughput applications, where a fast and efficient single-step approach would be desirable for obtaining coatings.

Nature's combinatorial biosynthesis and recently engineered production of nucleoside antibiotics in Streptomyces.

Modified nucleosides produced by Streptomyces and related actinomycetes are widely used in agriculture and medicine as antibacterial, antifungal, anticancer and antiviral agents. These specialized small-molecule metabolites are biosynthesized by complex enzymatic machineries encoded within gene clusters in the genome. The past decade has witnessed a burst of reports defining the key metabolic processes involved in the biosynthesis of several distinct families of nucleoside antibiotics. Furthermore, genome sequencing of various Streptomyces species has dramatically increased over recent years. Potential biosynthetic gene clusters for novel nucleoside antibiotics are now apparent by analysis of these genomes. Here we revisit strategies for production improvement of nucleoside antibiotics that have defined mechanisms of action, and are in clinical or agricultural use. We summarize the progress for genetically manipulating biosynthetic pathways for structural diversification of nucleoside antibiotics. Microorganism-based biosynthetic examples are provided and organized under genetic principles and metabolic engineering guidelines. We show perspectives on the future of combinatorial biosynthesis, and present a working model for discovery of novel nucleoside natural products in Streptomyces.

Direct Formation of Oxocarbenium Ions under Weakly Acidic Conditions: Catalytic Enantioselective Oxa-Pictet-Spengler Reactions.

Two catalysts, an amine HCl salt and a bisthiourea, work in concert to enable the generation of oxocarbenium ions under mild conditions. The amine catalyst generates an iminium ion of sufficient electrophilicity to enable 1,2-attack by an alcohol. Catalyst turnover is achieved by amine elimination with concomitant formation of an oxocarbenium intermediate. The bisthiourea catalyst accelerates all of the steps of the reaction and controls the stereoselectivity via anion binding/ion pair formation. This new concept was applied to direct catalytic enantioselective oxa-Pictet-Spengler reactions of tryptophol with aldehydes.

A Branch Point of Streptomyces Sulfur Amino Acid Metabolism Controls the Production of Albomycin.

Albomycin (ABM), also known as grisein, is a sulfur-containing metabolite produced by Streptomyces griseus ATCC 700974. Genes predicted to be involved in the biosynthesis of ABM and ABM-like molecules are found in the genomes of other actinomycetes. ABM has potent antibacterial activity, and as a result, many attempts have been made to develop ABM into a drug since the last century. Although the productivity of S. griseus can be increased with random mutagenesis methods, understanding of Streptomyces sulfur amino acid (SAA) metabolism, which supplies a precursor for ABM biosynthesis, could lead to improved and stable production. We previously characterized the gene cluster (abm) in the genome-sequenced S. griseus strain and proposed that the sulfur atom of ABM is derived from either cysteine (Cys) or homocysteine (Hcy). The gene product, AbmD, appears to be an important link between primary and secondary sulfur metabolic pathways. Here, we show that propargylglycine or iron supplementation in growth media increased ABM production by significantly changing the relative concentrations of intracellular Cys and Hcy. An SAA metabolic network of S. griseus was constructed. Pathways toward increasing Hcy were shown to positively impact ABM production. The abmD gene and five genes that increased the Hcy/Cys ratio were assembled downstream of hrdBp promoter sequences and integrated into the chromosome for overexpression. The ABM titer of one engineered strain, SCAK3, in a chemically defined medium was consistently improved to levels ∼400% of the wild type. Finally, we analyzed the production and growth of SCAK3 in shake flasks for further process development.

Analysis of the biosynthesis of antibacterial cyclic dipeptides in Nocardiopsis alba.

Nocardiopsis alba is frequently isolated from environment and has recently been suggested as a casual symbiotic actinobacterium of diverse invertebrates. Using activity-guided fractionation, we purified two antibacterial cyclic dipeptides, cyclo(ΔPhe-ΔLeu) (albonoursin) and cyclo(ΔmTyr-ΔLeu), from a culture of Nocardiopsis alba ATCC BAA-2165. Analysis of N. alba genome revealed genetic information similar to albonoursin biosynthetic gene cluster, albABC. An albABC gene deletion mutant of N. alba was generated. Liquid chromatography-mass spectrometry analysis showed that the mutant could not produce the cyclic dipeptides. Cyclic dipeptide production in the mutant was restored by genetic complementation with the albABC cloned in a native plasmid of Nocardiopsis. ß-Glucuronidase reporter assays with a second mutant construct, in which albABC promoter is transcriptionally fused to the reporting gene gusA, indicated that albABC gene expression was subject to osmoregulation. The system presented will be used to study the metabolic and genetic control of cyclic dipeptide biosynthesis in Nocardiopsis.

A streamlined process for discovery and characterization of inhibitors against phenylalanyl-tRNA synthetase of Mycobacterium tuberculosis.

Aminoacyl-tRNA synthetases (aaRSs) catalyze aminoacylation of tRNAs to produce aminoacyl-tRNAs for protein synthesis. Bacterial aaRSs have distinctive features, play an essential role in channeling amino acids into biomolecular assembly, and are vulnerable to inhibition by small molecules. The aaRSs continue to be targets for potential antibacterial drug development. The first step of aaRS reaction is the activation of amino acid by hydrolyzing ATP to form an acyladenylate intermediate with the concomitant release of pyrophosphate. None-radioactive assays usually measure the rate of ATP consumption or phosphate generation, offering advantages in high-throughput drug screening. These simple aaRS enzyme assays can be adapted to study the mode of inhibition of natural or synthetic aaRS inhibitors. Taking phenylalanyl-tRNA synthetase (PheRS) of Mycobacterium tuberculosis (Mtb) as an example, we describe a process for identification and characterization of Mtb PheRS inhibitor.

The Projectile Perforation Resistance of Materials: Scaling the Impact Resistance of Thin Films to Macroscale Materials.

From drug delivery to ballistic impact, the ability to control or mitigate the puncture of a fast-moving projectile through a material is critical. While puncture is a common occurrence, which can span many orders of magnitude in the size, speed, and energy of the projectile, there remains a need to connect our understanding of the perforation resistance of materials at the nano- and microscale to the actual behavior at the macroscale that is relevant for engineering applications. In this article, we address this challenge by combining a new dimensional analysis scheme with experimental data from micro- and macroscale impact tests to develop a relationship that connects the size-scale effects and materials properties during high-speed puncture events. By relating the minimum perforation velocity to fundamental material properties and geometric test conditions, we provide new insights and establish an alternative methodology for evaluating the performance of materials that is independent of the impact energy or the specific projectile puncture experiment type. Finally, we demonstrate the utility of this approach by assessing the relevance of novel materials, such as nanocomposites and graphene for real-world impact applications.

Whole-genome sequence of Nocardiopsis alba strain ATCC BAA-2165, associated with honeybees.

The actinomycete Nocardiopsis alba was reportedly associated with honeybees in separate occurrences. We report the complete genome of Nocardiopsis alba ATCC BAA-2165 isolated from honeybee guts. It will provide insights into the metabolism and genetic regulatory networks of this genus of bacteria that enable them to live in a range of environments.

A Computationally Efficient Approach to Simulate Heart Rate Effects Using a Whole Human Heart Model.

Computational modeling of the whole human heart has become a valuable tool to evaluate medical devices such as leadless pacemakers, annuloplasty rings and left ventricular assist devices, since it is often difficult to replicate the complex dynamic interactions between the device and human heart in bench-top and animal tests. The Dassault Systèmes Living Heart Human Model (LHHM) is a finite-element model of whole-human-heart electromechanics that has input parameters that were previously calibrated to generate physiological responses in a healthy heart beating at 60 beat/min (resting state). This study demonstrates that, by adjusting only six physiologically meaningful parameters, the LHHM can be recalibrated to generate physiological responses in a healthy heart beating at heart rates ranging from 90−160 beat/min. These parameters are as follows: the sinoatrial node firing period decreases from 0.67 s at 90 bpm to 0.38 s at 160 bpm, atrioventricular delay decreases from 0.122 s at 90 bpm to 0.057 s at 160 bpm, preload increases 3-fold from 90 bpm to 160 bpm, body resistance at 160 bpm is 80% of that at 90 bpm, arterial stiffness at 160 bpm is 3.9 times that at 90 bpm, and a parameter relating myofiber twitch force duration and sarcomere length decreases from 238 ms/mm at 90 bpm to 175 ms/mm at 160 bpm. In addition, this study demonstrates the feasibility of using the LHHM to conduct clinical investigations in AV delay optimization and hemodynamic differences between pacing and exercise. AV delays in the ranges of 40 ms to 250 ms were simulated and stroke volume and systolic blood pressure showed clear peaks at 120 ms for 90 bpm. For a heart during exercise, the increase in cardiac output continues to 160 bpm. However, for a heart during pacing, those physiological parameter adjustments are removed that are related to changes in body oxygen requirements (preload, arterial stiffness and body resistance). Consequently, cardiac output increases initially with heart rate; as the heart rate goes up (>100 bpm), the increasing rate of cardiac output slows down and approaches a plateau.

Investigate Natural Product Indolmycin and the Synthetically Improved Analogue Toward Antimycobacterial Agents.

Indolmycin (IND) is a microbial natural product that selectively inhibits bacterial tryptophanyl-tRNA synthetase (TrpRS). The tryptophan biosynthesis pathway was recently shown to be an important target for developing new antibacterial agents against Mycobacterium tuberculosis (Mtb). We investigated the antibacterial activity of IND against several mycobacterial model strains. A TrpRS biochemical assay was developed to analyze a library of synthetic IND analogues. The 4″-methylated IND compound, Y-13, showed improved anti-Mtb activity with a minimum inhibitory concentration (MIC) of 1.88 µM (∼0.5 µg/mL). The MIC increased significantly when overexpression of TrpRS was induced in the genetically engineered surrogate M. bovis BCG. The cocrystal structure of Mtb TrpRS complexed with IND and ATP has revealed that the amino acid pocket is in a state between the open form of apo protein and the closed complex with the reaction intermediate. In whole-cell-based experiments, we studied the combination effect of Y-13 paired with different antibacterial agents. We evaluated the killing kinetics, the frequency of resistance to INDs, and the mode of resistance of IND-resistant mycobacteria by genome sequencing. The synergistic interaction of Y-13 with the TrpE allosteric inhibitor, indole propionic acid, suggests that prospective IND analogues could shut down tryptophan biosynthesis and protein biosynthesis in pathogens, leading to a new class of antibiotics. Finally, we discuss a strategy to expand the genome mining of antibiotic-producing microbes specifically for antimycobacterial development.

Human Kinase IGF1R/IR Inhibitor Linsitinib Controls the In Vitro and Intracellular Growth of Mycobacterium tuberculosis.

ATP provides energy in the biosynthesis of cellular metabolites as well as regulates protein functions through phosphorylation. Many ATP-dependent enzymes are antibacterial and anticancer targets including human kinases acted on by most of the successful drugs. In search of new chemotherapeutics for tuberculosis (TB), we screened repurposing compounds against the essential glutamine synthase (GlnA1) of Mycobacterium tuberculosis (Mtb) and identified linsitinib, a clinical-stage drug originally targeting kinase IGF1R/IR as a potent GlnA1 inhibitor. Linsitinib has direct antimycobacterial activity. Biochemical, molecular modeling, and target engagement analyses revealed the inhibition is ATP-competitive and specific in Mtb. Linsitinib also improves autophagy flux in both Mtb-infected and uninfected THP1 macrophages, as demonstrated by the decreased p-mTOR and p62 and the increased lipid-bound LC3B-II and autophagosome forming puncta. Linsitinib-mediated autophagy reduces intracellular growth of wild-type and isoniazid-resistant Mtb alone or in combination with bedaquiline. We have demonstrated that an IGF-IR/IR inhibitor can potentially be used to treat TB. Our study reinforces the concept of targeting ATP-dependent enzymes for novel anti-TB therapy.

Inhibition of selenocysteine tRNA[Ser]Sec aminoacylation provides evidence that aminoacylation is required for regulatory methylation of this tRNA.

There are two isoforms of selenocysteine (Sec) tRNA([Ser]Sec) that differ by a single methyl group, Um34. The non-Um34 isoform supports the synthesis of a subclass of selenoproteins, designated housekeeping, while the Um34 isoform supports the expression of another subclass, designated stress-related selenoproteins. Herein, we investigated the relationship between tRNA([Ser]Sec) aminoacylation and Um34 synthesis which is the last step in the maturation of this tRNA. Mutation of the discriminator base at position 73 in tRNA([Ser]Sec) dramatically reduced aminoacylation with serine, as did an inhibitor of seryl-tRNA synthetase, SB-217452. Although both the mutation and the inhibitor prevented Um34 synthesis, neither precluded the synthesis of any other of the known base modifications on tRNA([Ser]Sec) following microinjection and incubation of the mutant tRNA([Ser]Sec) transcript, or the wild type transcript along with inhibitor, in Xenopus oocytes. The data demonstrate that Sec tRNA([Ser]Sec) must be aminoacylated for Um34 addition. The fact that selenium is required for Um34 methylation suggests that Sec must be attached to its tRNA for Um34 methylation. This would explain why selenium is essential for the function of Um34 methylase and provides further insights into the hierarchy of selenoprotein expression.

Biodistribution of neural stem cells after intravascular therapy for hypoxic-ischemia.

BACKGROUND AND PURPOSE: Intravascular transplantation of neural stem cells represents a minimally invasive therapeutic approach for the treatment of central nervous system diseases. The cellular biodistribution after intravascular injection needs to be analyzed to determine the ideal delivery modality. We studied the biodistribution and efficiency of targeted central nervous system delivery comparing intravenous and intra-arterial (IA) administration of neural stem cells after brain ischemia. METHODS: Mouse neural stem cells were transduced with a firefly luciferase reporter gene for bioluminescence imaging (BLI). Hypoxic-ischemia was induced in adult mice and reporter neural stem cells were transplanted IA or intravenous at 24 hours after brain ischemia. In vivo BLI was used to track transplanted cells up to 2 weeks after transplantation and ex vivo BLI was used to determine single organ biodistribution. RESULTS: Immediately after transplantation, BLI signal from the brain was 12 times higher in IA versus intravenous injected animals (P<0.0001). After IA injection, 69% of the total luciferase activity arose from the brain early after transplantation and 93% at 1 week. After intravenous injection, 94% of the BLI signal was detected in the lungs (P=0.004) followed by an overall 94% signal loss at 1 week, indicating lack of cell survival outside the brain. Ex vivo single organ analysis showed a significantly higher BLI signal in the brain than in the lungs, liver, and kidneys at 1 week (P<0.0001) and 2 weeks in IA (P=0.007). CONCLUSIONS: IA transplantation results in superior delivery and sustained presence of neural stem cells in the ischemic brain in comparison to intravenous infusion.

Characterization of two seryl-tRNA synthetases in albomycin-producing Streptomyces sp. strain ATCC 700974.

The Trojan horse antibiotic albomycin, produced by Streptomyces sp. strain ATCC 700974, contains a thioribosyl nucleoside moiety linked to a hydroxamate siderophore through a serine residue. The seryl nucleoside structure (SB-217452) is a potent inhibitor of seryl-tRNA synthetase (SerRS) in the pathogenic bacterium Staphylococcus aureus, with a 50% inhibitory concentration (IC(50)) of approximately 8 nM. In the albomycin-producing Streptomyces sp., a bacterial SerRS homolog (Alb10) was found to be encoded in a biosynthetic gene cluster in addition to another serRS gene (serS1) at a different genetic locus. Alb10, named SerRS2 herein, is significantly divergent from SerRS1, which shows high homology to the housekeeping SerRS found in other Streptomyces species. We genetically and biochemically characterized the two genes and the proteins encoded. Both genes were able to complement a temperature-sensitive serS mutant of Escherichia coli and allowed growth at a nonpermissive temperature. serS2 was shown to confer albomycin resistance, with specific amino acid residues in the motif 2 signature sequences of SerRS2 playing key roles. SerRS1 and SerRS2 are comparably efficient in vitro, but the K(m) of serine for SerRS2 measured during tRNA aminoacylation is more than 20-fold higher than that for SerRS1. SB-217452 was also enzymatically generated and purified by two-step chromatography. Its IC(50) against SerRS1 was estimated to be 10-fold lower than that against SerRS2. In contrast, both SerRSs displayed comparable inhibition kinetics for serine hydroxamate, indicating that SerRS2 was specifically resistant to SB-217452. These data suggest that mining Streptomyces genomes for duplicated aminoacyl-tRNA synthetase genes could provide a novel approach for the identification of natural products targeting aminoacyl-tRNA synthetases.