ABSTRACT
Splice site recognition is essential for defining the transcriptome. Drugs like risdiplam and branaplam change how U1 snRNP recognizes particular 5' splice sites (5'SS) and promote U1 snRNP binding and splicing at these locations. Despite the therapeutic potential of 5'SS modulators, the complexity of their interactions and snRNP substrates have precluded defining a mechanism for 5'SS modulation. We have determined a sequential binding mechanism for modulation of -1A bulged 5'SS by branaplam using a combination of ensemble kinetic measurements and colocalization single molecule spectroscopy (CoSMoS). Our mechanism establishes that U1-C protein binds reversibly to U1 snRNP, and branaplam binds to the U1 snRNP/U1-C complex only after it has engaged a -1A bulged 5'SS. Obligate orders of binding and unbinding explain how reversible branaplam interactions cause formation of long-lived U1 snRNP/5'SS complexes. Branaplam is a ribonucleoprotein, not RNA duplex alone, targeting drug whose action depends on fundamental properties of 5'SS recognition.
ABSTRACT
Nearly 30% of patients with relapsed breast cancer present activating mutations in estrogen receptor alpha (ERα) that confer partial resistance to existing endocrine-based therapies. We previously reported the development of H3B-5942, a covalent ERα antagonist that engages cysteine-530 (C530) to achieve potency against both wild-type (ERαWT) and mutant ERα (ERαMUT). Anticipating that the emergence of C530 mutations could promote resistance to H3B-5942, we applied structure-based drug design to improve the potency of the core scaffold to further enhance the antagonistic activity in addition to covalent engagement. This effort led to the development of the clinical candidate H3B-6545, a covalent antagonist that is potent against both ERαWT/MUT, and maintains potency even in the context of ERα C530 mutations. H3B-6545 demonstrates significant activity and superiority over standard-of-care fulvestrant across a panel of ERαWT and ERαMUT palbociclib sensitive and resistant models. In summary, the compelling preclinical activity of H3B-6545 supports its further development for the potential treatment of endocrine therapy-resistant ERα+ breast cancer harboring wild-type or mutant ESR1, as demonstrated by the ongoing clinical trials (NCT03250676, NCT04568902, NCT04288089). SUMMARY: H3B-6545 is an ERα covalent antagonist that exhibits encouraging preclinical activity against CDK4/6i naïve and resistant ERαWT and ERαMUT tumors.
Subject(s)
Breast Neoplasms , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Clinical Trials as Topic , Estrogen Receptor alpha/genetics , Female , Fulvestrant/therapeutic use , Humans , Indazoles , Neoplasm Recurrence, Local , PyridinesABSTRACT
The production of a mature mRNA requires coordination of multiple processing steps, which ultimately control its content, localization, and stability. These steps include some of the largest macromolecular machines in the cell, which were, until recently, considered undruggable due to their biological complexity. Building from an expanded understanding of the underlying mechanisms that drive these processes, a new wave of therapeutics is seeking to target RNA processing. With a focus on impacting gene regulation at the RNA level, such modalities offer potential for sequence-specific resolution in drug design. Here, we review our current understanding of RNA-processing events and their role in gene regulation, with a focus on the therapeutic opportunities that have emerged within this landscape.
Subject(s)
Oligonucleotides, Antisense , RNA Processing, Post-Transcriptional , Gene Expression Regulation , Humans , Oligonucleotides, Antisense/therapeutic use , RNA/genetics , RNA, MessengerABSTRACT
Carbamoyl phosphate synthetase 1 (CPS1) is a potential synthetic lethal target in LKB1-deficient nonsmall cell lung cancer, where its overexpression supports the production of pyrimidine synthesis. In other cancer types, CPS1 overexpression and activity may prevent the accumulation of toxic levels of intratumoral ammonia to support tumor growth. Herein we report the discovery of a novel series of potent and selective small-molecule inhibitors of CPS1. Piperazine 2 was initially identified as a promising CPS1 inhibitor through a high-throughput screening effort. Subsequent structure-activity relationship optimization and structure-based drug design led to the discovery of piperazine H3B-616 (25), a potent allosteric inhibitor of CPS1 (IC50 = 66 nM).
ABSTRACT
Carbamoyl phosphate synthetase 1 (CPS1) catalyzes the first step in the ammonia-detoxifying urea cycle, converting ammonia to carbamoyl phosphate under physiologic conditions. In cancer, CPS1 overexpression supports pyrimidine synthesis to promote tumor growth in some cancer types, while in others CPS1 activity prevents the buildup of toxic levels of intratumoral ammonia to allow for sustained tumor growth. Targeted CPS1 inhibitors may, therefore, provide a therapeutic benefit for cancer patients with tumors overexpressing CPS1. Herein, we describe the discovery of small-molecule CPS1 inhibitors that bind to a previously unknown allosteric pocket to block ATP hydrolysis in the first step of carbamoyl phosphate synthesis. CPS1 inhibitors are active in cellular assays, blocking both urea synthesis and CPS1 support of the pyrimidine biosynthetic pathway, while having no activity against CPS2. These newly discovered CPS1 inhibitors are a first step toward providing researchers with valuable tools for probing CPS1 cancer biology.
Subject(s)
Carbamoyl-Phosphate Synthase (Ammonia)/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Piperidines/pharmacology , Small Molecule Libraries/pharmacology , Thiazoles/pharmacology , Adenosine Triphosphate/antagonists & inhibitors , Adenosine Triphosphate/metabolism , Allosteric Regulation/drug effects , Carbamoyl-Phosphate Synthase (Ammonia)/genetics , Carbamoyl-Phosphate Synthase (Ammonia)/metabolism , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Humans , Hydrolysis/drug effects , Models, Molecular , Molecular Structure , Piperidines/chemistry , Small Molecule Libraries/chemistry , Thiazoles/chemistryABSTRACT
DNMT3A (DNA methyltransferase 3A) is a de novo DNA methyltransferase responsible for establishing CpG methylation patterns within the genome. DNMT3A activity is essential for normal development, and its dysfunction has been linked to developmental disorders and cancer. DNMT3A is frequently mutated in myeloid malignancies with the majority of mutations occurring at Arg-882, where R882H mutations are most frequent. The R882H mutation causes a reduction in DNA methyltransferase activity and hypomethylation at differentially-methylated regions within the genome, ultimately preventing hematopoietic stem cell differentiation and leading to leukemogenesis. Although the means by which the R882H DNMT3A mutation reduces enzymatic activity has been the subject of several studies, the precise mechanism by which this occurs has been elusive. Herein, we demonstrate that in the context of the full-length DNMT3A protein, the R882H mutation stabilizes the formation of large oligomeric DNMT3A species to reduce the overall DNA methyltransferase activity of the mutant protein as well as the WT-R882H complex in a dominant-negative manner. This shift in the DNMT3A oligomeric equilibrium and the resulting reduced enzymatic activity can be partially rescued in the presence of oligomer-disrupting DNMT3L, as well as DNMT3A point mutations along the oligomer-forming interface of the catalytic domain. In addition to modulating the oligomeric state of DNMT3A, the R882H mutation also leads to a DNA-binding defect, which may further reduce enzymatic activity. These findings provide a mechanistic explanation for the observed loss of DNMT3A activity associated with the R882H hot spot mutation in cancer.
Subject(s)
DNA (Cytosine-5-)-Methyltransferases/chemistry , DNA (Cytosine-5-)-Methyltransferases/metabolism , Mutation , Protein Multimerization , DNA/metabolism , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA Methyltransferase 3A , Humans , Models, Molecular , Protein Structure, QuaternaryABSTRACT
Mutations in estrogen receptor alpha (ERα) that confer resistance to existing classes of endocrine therapies are detected in up to 30% of patients who have relapsed during endocrine treatments. Because a significant proportion of therapy-resistant breast cancer metastases continue to be dependent on ERα signaling, there remains a critical need to develop the next generation of ERα antagonists that can overcome aberrant ERα activity. Through our drug-discovery efforts, we identified H3B-5942, which covalently inactivates both wild-type and mutant ERα by targeting Cys530 and enforcing a unique antagonist conformation. H3B-5942 belongs to a class of ERα antagonists referred to as selective estrogen receptor covalent antagonists (SERCA). In vitro comparisons of H3B-5942 with standard-of-care (SoC) and experimental agents confirmed increased antagonist activity across a panel of ERαWT and ERαMUT cell lines. In vivo, H3B-5942 demonstrated significant single-agent antitumor activity in xenograft models representing ERαWT and ERαY537S breast cancer that was superior to fulvestrant. Lastly, H3B-5942 potency can be further improved in combination with CDK4/6 or mTOR inhibitors in both ERαWT and ERαMUT cell lines and/or tumor models. In summary, H3B-5942 belongs to a class of orally available ERα covalent antagonists with an improved profile over SoCs.Significance: Nearly 30% of endocrine therapy-resistant breast cancer metastases harbor constitutively activating mutations in ERα. SERCA H3B-5942 engages C530 of both ERαWT and ERαMUT, promotes a unique antagonist conformation, and demonstrates improved in vitro and in vivo activity over SoC agents. Importantly, single-agent efficacy can be further enhanced by combining with CDK4/6 or mTOR inhibitors. Cancer Discov; 8(9); 1176-93. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 1047.
Subject(s)
Breast Neoplasms/drug therapy , Drug Resistance, Neoplasm/drug effects , Estrogen Receptor Antagonists/administration & dosage , Estrogen Receptor alpha/antagonists & inhibitors , Indazoles/administration & dosage , Mutation , Administration, Oral , Animals , Breast Neoplasms/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cysteine/antagonists & inhibitors , Drug Screening Assays, Antitumor , Drug Synergism , Estrogen Receptor Antagonists/chemistry , Estrogen Receptor Antagonists/pharmacology , Estrogen Receptor alpha/chemistry , Estrogen Receptor alpha/genetics , Female , Humans , Indazoles/chemistry , Indazoles/pharmacology , MCF-7 Cells , Mice , Protein Conformation/drug effects , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
Human rhinovirus (HRV) is the predominant cause of the common cold, but more importantly, infection may have serious repercussions in asthmatics and chronic obstructive pulmonary disorder (COPD) patients. A cell-based antiviral screen against HRV was performed with a subset of our proprietary compound collection, and an aminothiazole series with pan-HRV species and enteroviral activity was identified. The series was found to act at the level of replication in the HRV infectious cycle. In vitro selection and sequencing of aminothiazole series-resistant HRV variants revealed a single-nucleotide mutation leading to the amino acid change I42V in the essential HRV 3A protein. This same mutation has been previously implicated in resistance to enviroxime, a former clinical-stage antipicornavirus agent. Enviroxime-like compounds have recently been shown to target the lipid kinase phosphatidylinositol 4-kinase III beta (PI4KIIIß). A good correlation between PI4KIIIß activity and HRV antiviral potency was found when analyzing the data over 80 compounds of the aminothiazole series, covering a 750-fold potency range. The mechanism of action through PI4KIIIß inhibition was further demonstrated by small interfering RNA (siRNA) knockdown of PI4KB, which reduced HRV replication and also increased the potency of the PI4KIIIß inhibitors. Inhibitors from two different structural classes with promising pharmacokinetic profiles and with very good selectivity for PI4KIIIß were used to dissociate compound-related toxicity from target-related toxicity. Mortality was seen in all dosing groups of mice treated with either compound, therefore suggesting that short-term inhibition of PI4KIIIß is deleterious.
Subject(s)
1-Phosphatidylinositol 4-Kinase/antagonists & inhibitors , Cephalosporins/pharmacology , Rhinovirus/drug effects , Rhinovirus/enzymology , Thiazoles/pharmacology , 1-Phosphatidylinositol 4-Kinase/genetics , 1-Phosphatidylinositol 4-Kinase/metabolism , Animals , Antiviral Agents/pharmacology , Benzimidazoles/pharmacology , Cell Line, Tumor , Common Cold/drug therapy , Common Cold/virology , Female , HeLa Cells , Humans , Mice , Oximes , Polymorphism, Single Nucleotide , RNA Interference , RNA, Small Interfering , Rhinovirus/growth & development , Sulfonamides , Virus Replication/drug effects , Virus Replication/geneticsABSTRACT
Phosphatidylinositol-4-kinase IIIα (PI4KIIIα) is an essential host cell factor for hepatitis C virus (HCV) replication. An N-terminally truncated 130-kDa form was used to reconstitute an in vitro biochemical lipid kinase assay that was optimized for small-molecule compound screening and identified potent and specific inhibitors. Cell culture studies with PI4KIIIα inhibitors demonstrated that the kinase activity was essential for HCV RNA replication. Two PI4KIIIα inhibitors were used to select cell lines harboring HCV replicon mutants with a 20-fold loss in sensitivity to the compounds. Reverse genetic mapping isolated an NS4B-NS5A segment that rescued HCV RNA replication in PIK4IIIα-deficient cells. HCV RNA replication occurs on specialized membranous webs, and this study with PIK4IIIα inhibitor-resistant mutants provides a genetic link between NS4B/NS5A functions and PI4-phosphate lipid metabolism. A comprehensive assessment of PI4KIIIα as a drug target included its evaluation for pharmacologic intervention in vivo through conditional transgenic murine lines that mimic target-specific inhibition in adult mice. Homozygotes that induce a knockout of the kinase domain or knock in a single amino acid substitution, kinase-defective PI4KIIIα, displayed a lethal phenotype with a fairly widespread mucosal epithelial degeneration of the gastrointestinal tract. This essential host physiologic role raises doubt about the pursuit of PI4KIIIα inhibitors for treatment of chronic HCV infection.
Subject(s)
1-Phosphatidylinositol 4-Kinase/metabolism , Hepacivirus/physiology , Host-Pathogen Interactions , Virus Replication , 1-Phosphatidylinositol 4-Kinase/antagonists & inhibitors , Animals , Antiviral Agents/pharmacology , Cell Line , DNA Mutational Analysis , Drug Resistance, Viral , Enzyme Inhibitors/pharmacology , Female , Genes, Essential , Hepatocytes/enzymology , Hepatocytes/virology , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Mutant Proteins/genetics , Viral Nonstructural Proteins/geneticsABSTRACT
The HCV p7 protein is not involved in viral RNA replication but is essential for production of infectious virus. Based on its putative ion channel activity, p7 belongs to a family of viral proteins known as viroporins that oligomerize after insertion into a lipid membrane. To screen for compounds capable of interfering with p7 channel function, a low-throughput liposome-based fluorescent dye permeability assay was modified and converted to a robust high-throughput screening assay. Escherichia coli expressing recombinant p7 were grown in high-density fed-batch fermentation followed by a detergent-free purification using a combination of affinity and reversed-phase chromatography. The phospholipid composition of the liposomes was optimized for both p7 recognition and long-term stability. A counterscreen was developed using the melittin channel-forming peptide to eliminate nonspecific screening hits. The p7 liposome-based assay displayed robust statistics (Z' > 0.75), and sensitivity to inhibition was confirmed using known inhibitors.
Subject(s)
Drug Evaluation, Preclinical/methods , High-Throughput Screening Assays , Ion Channels/metabolism , Recombinant Proteins/metabolism , Viral Proteins/metabolism , Chromatography, Liquid , Humans , Ion Channels/genetics , Ion Channels/isolation & purification , Liposomes/chemistry , Liposomes/metabolism , Melitten/metabolism , Permeability , Phospholipids/chemistry , Phospholipids/metabolism , Protein Stability , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Sensitivity and Specificity , Small Molecule Libraries , Viral Proteins/genetics , Viral Proteins/isolation & purificationABSTRACT
A functional screen of an adenovirus-delivered shRNA library that targets approximately 4500 host genes was performed to identify cellular factors that regulate hepatitis C virus (HCV) sub-genomic RNA replication. Seventy-three hits were further examined by siRNA oligonucleotide-directed knockdown, and silencing of the PI4KA gene was demonstrated to have a significant effect on the replication of a HCV genotype 1b replicon. Using transient siRNA oligonucleotide transfections and stable shRNA knockdown clones in HuH-7 cells, the PI4KA gene was shown to be essential for the replication of all HCV genotypes tested (1a, 1b and 2a) but not required for bovine viral diarrhea virus (BVDV) RNA replication.
Subject(s)
Hepacivirus/physiology , Hepatitis C/enzymology , Phosphotransferases (Alcohol Group Acceptor)/metabolism , RNA, Viral/genetics , Virus Replication/genetics , Adenoviridae/genetics , Adenoviridae/metabolism , Adenoviridae/physiology , Cell Line, Tumor , Gene Expression Regulation, Viral , Gene Knockdown Techniques , Gene Library , Genome, Viral , Hepacivirus/genetics , Hepacivirus/growth & development , Hepacivirus/metabolism , Humans , Minor Histocompatibility Antigens , Phosphotransferases (Alcohol Group Acceptor)/genetics , RNA, Small Interfering/metabolism , Reproducibility of ResultsABSTRACT
Aliphatic halogenases activate O(2), cleave alpha-ketoglutarate (alphaKG) to CO(2) and succinate, and form haloferryl [X-Fe(IV)O; X = Cl or Br] complexes that cleave aliphatic C-H bonds to install halogens during the biosynthesis of natural products by non-ribosomal peptide synthetases (NRPSs). For the related alphaKG-dependent dioxygenases, it has been shown that reaction of the Fe(II) cofactor with O(2) to form the C-H bond-cleaving ferryl complex is "triggered" by binding of the target substrate. In this study, we have tested for and defined structural determinants of substrate triggering (ST) in the halogenase, SyrB2, from the syringomycin E biosynthetic NRPS of Pseudomonas syringae B301D. As for other halogenases, the substrate of SyrB2 is complex, consisting of l-Thr tethered via a thioester linkage to a covalently bound phosphopantetheine (PPant) cofactor of a carrier protein, SyrB1. Without an appended amino acid, SyrB1 does not trigger formation of the chloroferryl intermediate state in SyrB2, even in the presence of free l-Thr or its analogues, but SyrB1 charged either by l-Thr (l-Thr-S-SyrB1) or by any of several non-native amino acids does trigger the reaction by as much as 8000-fold (for the native substrate). Triggering efficacy is sensitive to the structures of both the amino acid and the carrier protein, being diminished by 5-24-fold when the native l-Thr is replaced with another amino acid and by approximately 40-fold when SyrB1 is replaced with the heterologous carrier protein, CytC2. The directing effect of the carrier protein and consequent tolerance for profound modifications to the target amino acid allow the chloroferryl state to be formed in the presence of substrates that perturb the ratio of its two putative coordination isomers, lack the target C-H bond (l-Ala-S-SyrB1), or contain a C-H bond of enhanced strength (l-cyclopropylglycyl-S-SyrB1). For the latter two cases, the SyrB2 chloroferryl state so formed exhibits unprecedented stability (t(1/2) = 30-110 min at 0 degree C), can be trapped at high concentration and purity by manual freezing without a cryosolvent, and represents an ideal target for structural characterization. As initial steps toward this goal, extended X-ray absorption fine structure (EXAFS) spectroscopy has been used to determine the Fe-O and Fe-Cl distances and density functional theory (DFT) calculations have been used to confirm that the measured distances are consistent with the anticipated structure of the intermediate.
Subject(s)
Carbon/chemistry , Hydrogen/chemistry , Oxidoreductases/chemistry , Catalysis , Crystallography, X-Ray , Kinetics , Molecular Structure , Oxygen/chemistry , Oxygenases/chemistry , Pseudomonas syringae/metabolism , Spectrophotometry/methods , Substrate Specificity , Temperature , Time FactorsABSTRACT
During the biosynthesis of the cyclopropyl amino acid coronamic acid from l-allo-Ile by the phytotoxic Pseudomonas syringae, the aminoacyl group covalently attached to the pantetheinyl arm of CmaA is shuttled to the HS-pantetheinyl arm of the protein CmaD by the aminoacyltransferase CmaE. CmaE will only recognize deacylated CmaA for initial complexation. The aminoacyl group becomes covalently attached to the active site Cys of CmaE and can then be transferred out to the holo pantetheinylated form of CmaD. Both l-Val/l-[14C]Val exchange studies and MALDI-TOF support a reversible shuttling process. Aminoacylated-S-CmaE will transfer the l-Val moiety to the HS-pantetheinyl arm of other T domains, including CytC2, BarA, and ArfA C2-A2-T2 but not to free HS-pantetheine. CmaD could be loaded with other amino acids, for example, l-Leu and l-Thr, by the action of heterologous donor T domains containing alternative aminoacyl groups. Additionally, CmaE is able to accept l-Phe as a substrate when presented on CmaD and is able to load this aminoacyl moiety onto heterologous T domains, expanding the potential for CmaE to be used as a tool for generating chemical diversity within an NRPS assembly line.
Subject(s)
Amino Acids/biosynthesis , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Biosynthetic Pathways , Amino Acid Motifs , Amino Acid Sequence , Aminoacylation , Bacterial Proteins/genetics , Histidine/chemistry , Isoleucine/metabolism , Kinetics , Models, Chemical , Molecular Sequence Data , Phenylalanine/chemistry , Protein Structure, Tertiary , Pseudomonas syringae/enzymology , Sequence Homology, Amino Acid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Substrate SpecificityABSTRACT
Syringomycin, a lipopeptidolactone assembled from nine amino acid monomers by four enzymes, SyrB1, SyrB2, SyrC, and SyrE, is a cyclic nonribosomal peptide made by plant-associated Pseudomonas spp. This assembly is unusual because the terminal residue, 4-chlorothreonine, has been proposed to be added in trans since the ninth module of the megasynthetase SyrE lacks an adenylation domain required for Thr/Cl-Thr activation. SyrC is now identified as a Thr/Cl-Thr aminoacyltransferase, shuttling the Thr/Cl-Thr moiety between the pantetheinyl arms of the thiolation domain of SyrB1 and the thiolation domain in module nine of SyrE. SyrC uses Cys224 as a catalytic nucleophile to generate a Thr/Cl-Thr-S-enzyme intermediate during transfer. SyrC joins a growing family of such aminoacyl-shuttling enzymes that also use covalent catalysis to move aminoacyl groups from carrier proteins during coumermycin and coronamic acid biosynthesis.
Subject(s)
Aminoacyltransferases/metabolism , Bacterial Proteins/biosynthesis , Bacterial Proteins/metabolism , Peptide Synthases/metabolism , Threonine/metabolism , Biological Transport , Pseudomonas syringae/enzymology , Threonine/analogs & derivativesABSTRACT
Four adjacent open reading frames, cytC1-C4, were cloned from a cytotrienin-producing strain of a Streptomyces sp. by using primers derived from the conserved region of a gene encoding a nonheme iron halogenase, CmaB, in coronamic acid biosynthesis. CytC1-3 were active after expression in Escherichia coli, and CytC4 was active after expression in Pseudomonas putida. CytC1, a relatively promiscuous adenylation enzyme, installs the aminoacyl moieties on the phosphopantetheinyl arm of the holo carrier protein CytC2. CytC3 is a nonheme iron halogenase that will generate both gamma-chloro- and gamma,gamma-dichloroaminobutyryl-S-CytC2 from aminobutyryl-S-CytC2. CytC4, a thioesterase, hydrolytically releases the dichloroaminobutyrate, a known streptomycete antibiotic. Thus, this short four-protein pathway is likely the biosynthetic source of this amino acid antimetabolite. This four-enzyme system analogously converts the proS-methyl group of valine to the dichloromethyl product regio- and stereospecifically.
Subject(s)
Antimetabolites/metabolism , Bacterial Proteins/metabolism , Butyrates/metabolism , Streptomyces/metabolism , Bacterial Proteins/genetics , Cloning, Molecular , Escherichia coli/metabolism , Multigene Family , Peptide Synthases/metabolism , Pseudomonas putida/metabolismABSTRACT
Ring-cleaving dioxygenases catalyze the oxygenolytic fission of catecholic compounds, a critical step in the aerobic degradation of aromatic compounds by bacteria. Two classes of these enzymes have been identified, based on the mode of ring cleavage: intradiol dioxygenases utilize non-heme Fe(III) to cleave the aromatic nucleus ortho to the hydroxyl substituents; and extradiol dioxygenases utilize non-heme Fe(II) or other divalent metal ions to cleave the aromatic nucleus meta to the hydroxyl substituents. Recent genomic, structural, spectroscopic, and kinetic studies have increased our understanding of the distribution, evolution, and mechanisms of these enzymes. Overall, extradiol dioxygenases appear to be more versatile than their intradiol counterparts. Thus, the former cleave a wider variety of substrates, have evolved on a larger number of structural scaffolds, and occur in a wider variety of pathways, including biosynthetic pathways and pathways that degrade non-aromatic compounds. The catalytic mechanisms of the two enzymes proceed via similar iron-alkylperoxo intermediates. The ability of extradiol enzymes to act on a variety of non-catecholic compounds is consistent with proposed differences in the breakdown of this iron-alkylperoxo intermediate in the two enzymes, involving alkenyl migration in extradiol enzymes and acyl migration in intradiol enzymes. Nevertheless, despite recent advances in our understanding of these fascinating enzymes, the major determinant of the mode of ring cleavage remains unknown.
Subject(s)
Dioxygenases/metabolism , Hydrocarbons, Aromatic/metabolism , Oxygen/metabolism , Oxygenases/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Catalysis , Dioxygenases/chemistry , Enzyme Activation , Hydrocarbons, Aromatic/chemistry , Models, Molecular , Oxidation-Reduction , Oxygenases/chemistrySubject(s)
Bacterial Proteins/chemistry , Bromine/chemistry , Carrier Proteins/chemistry , Chlorine/chemistry , Oxidoreductases/chemistry , Peptides, Cyclic/biosynthesis , Bacterial Proteins/metabolism , Carrier Proteins/metabolism , Chlorine/metabolism , Molecular Conformation , Oxidoreductases/metabolism , Peptides, Cyclic/chemistry , Pseudomonas syringae/enzymology , Pseudomonas syringae/metabolism , Time FactorsABSTRACT
Non-haem Fe(II)/alpha-ketoglutarate (alphaKG)-dependent enzymes harness the reducing power of alphaKG to catalyse oxidative reactions, usually the hydroxylation of unactivated carbons, and are involved in processes such as natural product biosynthesis, the mammalian hypoxic response, and DNA repair. These enzymes couple the decarboxylation of alphaKG with the formation of a high-energy ferryl-oxo intermediate that acts as a hydrogen-abstracting species. All previously structurally characterized mononuclear iron enzymes contain a 2-His, 1-carboxylate motif that coordinates the iron. The two histidines and one carboxylate, known as the 'facial triad', form one triangular side of an octahedral iron coordination geometry. A subclass of mononuclear iron enzymes has been shown to catalyse halogenation reactions, rather than the more typical hydroxylation reaction. SyrB2, a member of this subclass, is a non-haem Fe(II)/alphaKG-dependent halogenase that catalyses the chlorination of threonine in syringomycin E biosynthesis. Here we report the structure of SyrB2 with both a chloride ion and alphaKG coordinated to the iron ion at 1.6 A resolution. This structure reveals a previously unknown coordination of iron, in which the carboxylate ligand of the facial triad is replaced by a chloride ion.
Subject(s)
Bacterial Proteins/biosynthesis , Iron/metabolism , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Pseudomonas syringae/enzymology , Bacterial Proteins/chemistry , Binding Sites , Chlorides/metabolism , Crystallography, X-Ray , Histidine/metabolism , Ketoglutaric Acids/metabolism , Ligands , Models, Molecular , Protein Conformation , Pseudomonas syringae/classification , Pseudomonas syringae/metabolismABSTRACT
The in vitro reconstitution of leucine halogenation during barbamide biosynthesis has been accomplished. It has been demonstrated that the triple chlorination of the unactivated pro-R methyl group of the peptidyl carrier protein-tethered l-Leu substrate is carried out by the tandem action of two nonheme iron(II)-dependent halogenases, BarB1 and BarB2. Investigation of the substrate specificities of each of the halogenating enzymes revealed their complementary roles in the generation of trichloroleucine.