RESUMO
Isopentenyl phosphate kinases (IPKs) have recently garnered attention for their central role in biocatalytic "isoprenol pathways," which seek to reduce the synthesis of the isoprenoid precursors to two enzymatic steps. Furthermore, the natural promiscuity of IPKs toward non-natural alkyl-monophosphates (alkyl-Ps) as substrates has hinted at the isoprenol pathways' potential to access novel isoprenoids with potentially useful activities. However, only a handful of IPK crystal structures have been solved to date, and even fewer of these contain non-natural substrates bound in the active site. The current study sought to elucidate additional ternary complexes bound to non-natural substrates using the IPK homolog from Thermococcus paralvinellae (TcpIPK). Four such structures were solved, each bound to a different non-natural alkyl-P and the phosphoryl donor substrate/product adenosine triphosphate (ATP)/adenosine diphosphate (ADP). As expected, the quaternary, tertiary, and secondary structures of TcpIPK closely resembled those of IPKs published previously, and kinetic analysis of a novel alkyl-P substrate highlighted the potentially dramatic effects of altering the core scaffold of the natural substrate. Even more interesting, though, was the discovery of a trend correlating the position of two α helices in the active site with the magnitude of an IPK homolog's reaction rate for the natural reaction. Overall, the current structures of TcpIPK highlight the importance of continued structural analysis of the IPKs to better understand and optimize their activity with both natural and non-natural substrates.
Assuntos
Trifosfato de Adenosina , Domínio Catalítico , Thermococcus , Especificidade por Substrato , Thermococcus/enzimologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Cinética , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Proteínas Arqueais/genética , Hemiterpenos/metabolismo , Hemiterpenos/química , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Conformação Proteica em alfa-Hélice , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/química , Clonagem Molecular , Expressão Gênica , Conformação Proteica em Folha beta , Sequência de Aminoácidos , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/enzimologia , Proteínas QuinasesRESUMO
S-adenosyl-l-methionine (AdoMet) is an essential enzyme cosubstrate in fundamental biology with an expanding range of biocatalytic and therapeutic applications. We report the design, synthesis, and evaluation of stable, functional AdoMet isosteres that are resistant to the primary contributors to AdoMet degradation (depurination, intramolecular cyclization, and sulfonium epimerization). Corresponding biochemical and structural studies demonstrate the AdoMet surrogates to serve as competent enzyme cosubstrates and to bind a prototypical class I model methyltransferase (DnrK) in a manner nearly identical to AdoMet. Given this conservation in function and molecular recognition, the isosteres presented are anticipated to serve as useful surrogates in other AdoMet-dependent processes and may also be resistant to, and/or potentially even inhibit, other therapeutically relevant AdoMet-dependent metabolic transformations (such as the validated drug target AdoMet decarboxylase). This work also highlights the ability of the prototypical class I model methyltransferase DnrK to accept non-native surrogate acceptors as an enabling feature of a new high-throughput methyltransferase assay.
Assuntos
S-Adenosilmetionina/química , HidróliseRESUMO
Structure analysis and ensemble refinement of the apo-structure of thymidine diphosphate (TDP)-rhamnose 3'-O-methyltransferase reveal a gate for substrate entry and product release. TDP-rhamnose 3'-O-methyltransferase (CalS11) catalyses a 3'-O-methylation of TDP-rhamnose, an intermediate in the biosynthesis of enediyne antitumor antibiotic calicheamicin. CalS11 operates at the sugar nucleotide stage prior to glycosylation step. Here, we present the crystal structure of the apo form of CalS11 at 1.89 Å resolution. We propose that the L2 loop functions as a gate facilitating and/or providing specificity for substrate entry or promoting product release. Ensemble refinement analysis slightly improves the crystallographic refinement statistics and furthermore provides a compelling way to visualize the dynamic model of loop L2, supporting the understanding of its proposed role in catalysis.
RESUMO
AT2433 from Actinomadura melliaura is an indolocarbazole antitumor antibiotic structurally distinguished by its unique aminodideoxypentose-containing disaccharide moiety. The corresponding sugar nucleotide-based biosynthetic pathway for this unusual sugar derives from comparative genomics where AtmS13 has been suggested as the contributing sugar aminotransferase (SAT). Determination of the AtmS13 X-ray structure at 1.50-Å resolution reveals it as a member of the aspartate aminotransferase fold type I (AAT-I). Structural comparisons of AtmS13 with homologous SATs that act upon similar substrates implicate potential active site residues that contribute to distinctions in sugar C5 (hexose vs. pentose) and/or sugar C2 (deoxy vs. hydroxyl) substrate specificity.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Carbazóis/metabolismo , Transaminases/química , Transaminases/metabolismo , Actinomycetales/enzimologia , Actinomycetales/genética , Proteínas de Bactérias/genética , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transaminases/genéticaRESUMO
Calicheamicin γ1I (1) is an enediyne antitumor compound produced by Micromonospora echinospora spp. calichensis, and its biosynthetic gene cluster has been previously reported. Despite extensive analysis and biochemical study, several genes in the biosynthetic gene cluster of 1 remain functionally unassigned. Using a structural genomics approach and biochemical characterization, two proteins encoded by genes from the 1 biosynthetic gene cluster assigned as "unknowns", CalU16 and CalU19, were characterized. Structure analysis revealed that they possess the STeroidogenic Acute Regulatory protein related lipid Transfer (START) domain known mainly to bind and transport lipids and previously identified as the structural signature of the enediyne self-resistance protein CalC. Subsequent study revealed calU16 and calU19 to confer resistance to 1, and reminiscent of the prototype CalC, both CalU16 and CalU19 were cleaved by 1 in vitro. Through site-directed mutagenesis and mass spectrometry, we identified the site of cleavage in each protein and characterized their function in conferring resistance against 1. This report emphasizes the importance of structural genomics as a powerful tool for the functional annotation of unknown proteins.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana/genética , Enedi-Inos/farmacologia , Micromonospora/metabolismo , Proteínas de Bactérias/genética , Cristalografia por Raios X , Genômica/métodos , Lipídeos/química , Micromonospora/crescimento & desenvolvimento , Modelos Moleculares , Estrutura Molecular , Família Multigênica , Mutagênese Sítio-Dirigida , Mutação/genética , Estrutura Terciária de Proteína , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
A nanoporous membrane system with directed flow carrying reagents to sequentially attached enzymes to mimic nature's enzyme complex system was demonstrated. Genetically modified glycosylation enzyme, OleD Loki variant, was immobilized onto nanometer-scale electrodes at the pore entrances/exits of anodic aluminum oxide membranes through His6-tag affinity binding. The enzyme activity was assessed in two reactionsa one-step "reverse" sugar nucleotide formation reaction (UDP-Glc) and a two-step sequential sugar nucleotide formation and sugar nucleotide-based glycosylation reaction. For the one-step reaction, enzyme specific activity of 620 min(1) on membrane supports was seen to be comparable to solution enzyme specific activity of 10 min(1). UDP-Glc production efficiencies as high as 98% were observed at a flow rate of 0.5 mL/min, at which the substrate residence time over the electrode length down pore entrances was matched to the enzyme activity rate. This flow geometry also prevented an unwanted secondary product hydrolysis reaction, as observed in the test homogeneous solution. Enzyme utilization increased by a factor of 280 compared to test homogeneous conditions due to the continuous flow of fresh substrate over the enzyme. To mimic enzyme complex systems, a two-step sequential reaction using OleD Loki enzyme was performed at membrane pore entrances then exits. After UDP-Glc formation at the entrance electrode, aglycon 4-methylumbelliferone was supplied at the exit face of the reactor, affording overall 80% glycosylation efficiency. The membrane platform showed the ability to be regenerated with purified enzyme as well as directly from expression crude, thus demonstrating a single-step immobilization and purification process.
Assuntos
Óxido de Alumínio/química , Enzimas Imobilizadas/química , Membranas Artificiais , Biocatálise , Eletrodos , Enzimas Imobilizadas/metabolismo , Glucose/química , Glucosiltransferases/química , Glucosiltransferases/metabolismo , Glicosilação , Difosfato de Uridina/químicaRESUMO
Although bacterial iterative type I polyketide synthases are now known to participate in the biosynthesis of a small set of diverse natural products, the subsequent downstream modification of the resulting polyketide products is poorly understood. We report the functional characterization of the putative orsellinic acid C2-O-methyltransferase, which is involved in calicheamicin biosynthesis. This study suggests that C2-O-methylation precedes C3-hydroxylation/methylation and C5-iodination and requires a coenzyme A- or acyl carrier protein-bound substrate.
Assuntos
Bactérias/enzimologia , Metiltransferases/metabolismo , Resorcinóis/metabolismo , Bactérias/química , Bactérias/metabolismo , Hidroxilação , Cinética , Metilação , Modelos Moleculares , Policetídeo Sintases/metabolismo , Estrutura Terciária de Proteína , S-Adenosilmetionina/metabolismo , Streptomyces/química , Streptomyces/enzimologia , Streptomyces/metabolismo , Especificidade por SubstratoRESUMO
UNLABELLED: Methionine adenosyltransferase (MAT) is a family of enzymes that utilizes ATP and methionine to produce S-adenosylmethionine (AdoMet), the most crucial methyl donor in the biological methylation of biomolecules and bioactive natural products. Here, we report that the MAT from Sulfolobus solfataricus (sMAT), an enzyme from a poorly explored class of the MAT family, has the ability to produce a range of differentially alkylated AdoMet analogs in the presence of non-native methionine analogs and ATP. To investigate the molecular basis for AdoMet analog production, we have crystallized the sMAT in the AdoMet bound, S-adenosylethionine (AdoEth) bound and unbound forms. Notably, among these structures, the AdoEth bound form offers the first MAT structure containing a non-native product, and cumulatively these structures add new structural insight into the MAT family and allow for detailed active site comparison with its homologs in Escherichia coli and human. As a thermostable MAT structure from archaea, the structures herein also provide a basis for future engineering to potentially broaden AdoMet analog production as reagents for methyltransferase-catalyzed 'alkylrandomization' and/or the study of methylation in the context of biological processes. DATABASES: PDB IDs: 4HPV, 4L7I, 4K0B and 4L2Z. EC 2.5.1.6 STRUCTURED DIGITAL ABSTRACT: ⢠sMAT and sMAT bind by x-ray crystallography (View interaction).
Assuntos
Proteínas Arqueais/química , Metionina Adenosiltransferase/química , Sulfolobus solfataricus/enzimologia , Motivos de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Cinética , Metionina/química , Modelos Moleculares , Ligação Proteica , Estrutura Quaternária de Proteína , Especificidade por SubstratoRESUMO
A chemoenzymatic platform for the synthesis of S-adenosyl-L-methionine (SAM) analogues compatible with downstream SAM-utilizing enzymes is reported. Forty-four non-native S/Se-alkylated Met analogues were synthesized and applied to probing the substrate specificity of five diverse methionine adenosyltransferases (MATs). Human MATâ II was among the most permissive of the MATs analyzed and enabled the chemoenzymatic synthesis of 29 non-native SAM analogues. As a proof of concept for the feasibility of natural product "alkylrandomization", a small set of differentially-alkylated indolocarbazole analogues was generated by using a coupled hMAT2-RebM system (RebM is the sugar C4'-O-methyltransferase that is involved in rebeccamycin biosynthesis). The ability to couple SAM synthesis and utilization in a single vessel circumvents issues associated with the rapid decomposition of SAM analogues and thereby opens the door for the further interrogation of a wide range of SAM utilizing enzymes.
Assuntos
S-Adenosilmetionina/química , S-Adenosilmetionina/síntese química , Biocatálise , Humanos , Estrutura MolecularRESUMO
Venturicidin C (1), a new 20-membered macrolide along with the known venturicidins A (2) and B (3) were isolated from the crude extract of the Appalachian bacterial strain Streptomyces sp. TS-2-2. Additionally, nine other known compounds namely nocardamine, dehydroxynocardamine, desmethylenylnocardamine, ferrioxamine E, adenosine, riboflavin, cyclo(D)-trans-4-OH-Pro-(D)-Phe, cyclo(D)-Pro-(D)-Phe and N-(2-phenylethyl)-acetamide were also isolated and identified. The structure of the new macrolide 1 was elucidated by the cumulative analyses of NMR spectroscopy and HR-MS data. Complete NMR assignments for the known venturicidins A (2) and B (3) are also provided, for the first time, in this report. Venturicidins A-C did not inhibit the proliferation of A549 lung cancer cell line but all displayed potent antifungal activity.
Assuntos
Antifúngicos/farmacologia , Macrolídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Streptomyces/metabolismo , Antifúngicos/química , Antifúngicos/isolamento & purificação , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Macrolídeos/química , Macrolídeos/isolamento & purificação , Espectroscopia de Ressonância Magnética , Espectrometria de Massas/métodos , Peptídeos Cíclicos/química , Peptídeos Cíclicos/isolamento & purificaçãoRESUMO
Sugar methyltransferases (MTs) are an important class of tailoring enzymes that catalyze the transfer of a methyl group from S-adenosyl-l-methionine to sugar-based N-, C- and O-nucleophiles. While sugar N- and C-MTs involved in natural product biosynthesis have been found to act on sugar nucleotide substrates prior to a subsequent glycosyltransferase reaction, corresponding sugar O-methylation reactions studied thus far occur after the glycosyltransfer reaction. Herein we report the first in vitro characterization using (1)H-(13)C-gHSQC with isotopically labeled substrates and the X-ray structure determination at 1.55 Å resolution of the TDP-3'-O-rhamnose-methyltransferase CalS11 from Micromonospora echinospora. This study highlights a unique NMR-based methyltransferase assay, implicates CalS11 to be a metal- and general acid/base-dependent O-methyltransferase, and as a first crystal structure for a TDP-hexose-O-methyltransferase, presents a new template for mechanistic studies and/or engineering.
Assuntos
Aminoglicosídeos/biossíntese , Metiltransferases/química , Metiltransferases/metabolismo , Ramnose/química , Catálise , Domínio Catalítico , Enedi-Inos , Espectroscopia de Ressonância Magnética , Micromonospora/enzimologia , Modelos Moleculares , Estrutura MolecularRESUMO
The molecule known as SF2575 from Streptomyces sp. is a tetracycline polyketide natural product that displays antitumor activity against murine leukemia P388 in vivo. In the SF2575 biosynthetic pathway, SsfS6 has been implicated as the crucial C-glycosyltransferase (C-GT) that forms the C-C glycosidic bond between the sugar and the SF2575 tetracycline-like scaffold. Here, we report the crystal structure of SsfS6 in the free form and in complex with TDP, both at 2.4 Å resolution. The structures reveal SsfS6 to adopt a GT-B fold wherein the TDP and docked putative aglycon are consistent with the overall C-glycosylation reaction. As one of only a few existing structures for C-glycosyltransferases, the structures described herein may serve as a guide to better understand and engineer C-glycosylation.
Assuntos
Antibacterianos/administração & dosagem , Proteínas de Bactérias/química , Cristalografia por Raios X , Tetraciclinas/química , Animais , Glicosilação , Glicosiltransferases/biossíntese , Glicosiltransferases/química , Leucemia P388/tratamento farmacológico , Leucemia P388/metabolismo , Leucemia P388/patologia , Camundongos , Streptomyces/química , Streptomyces/metabolismo , Tetraciclinas/biossínteseRESUMO
Glycosyltransferases are useful synthetic catalysts for generating natural products with sugar moieties. Although several natural product glycosyltransferase structures have been reported, design principles of glycosyltransferase engineering for the generation of glycodiversified natural products has fallen short of its promise, partly due to a lack of understanding of the relationship between structure and function. Here, we report structures of all four calicheamicin glycosyltransferases (CalG1, CalG2, CalG3, and CalG4), whose catalytic functions are clearly regiospecific. Comparison of these four structures reveals a conserved sugar donor binding motif and the principles of acceptor binding region reshaping. Among them, CalG2 possesses a unique catalytic motif for glycosylation of hydroxylamine. Multiple glycosyltransferase structures in a single natural product biosynthetic pathway are a valuable resource for understanding regiospecific reactions and substrate selectivities and will help future glycosyltransferase engineering.
Assuntos
Aminoglicosídeos/biossíntese , Antibióticos Antineoplásicos/biossíntese , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Engenharia de Proteínas/métodos , Domínios e Motivos de Interação entre Proteínas/genética , Carboidratos/química , Enedi-Inos/química , Hidroxilaminas/metabolismoRESUMO
Mitomycins are quinone-containing antibiotics, widely used as antitumor drugs in chemotherapy. Mitomycin-7-O-methyltransferase (MmcR), a key tailoring enzyme involved in the biosynthesis of mitomycin in Streptomyces lavendulae, catalyzes the 7-O-methylation of both C9ß- and C9α-configured 7-hydroxymitomycins. We have determined the crystal structures of the MmcR-S-adenosylhomocysteine (SAH) binary complex and MmcR-SAH-mitomycin A (MMA) ternary complex at resolutions of 1.9and 2.3 Å, respectively. The study revealed MmcR to adopt a common S-adenosyl-L-methionine-dependent O-methyltransferase fold and the presence of a structurally conserved active site general acid-base pair is consistent with a proton-assisted methyltransfer common to most methyltransferases. Given the importance of C7 alkylation to modulate mitomycin redox potential, this study may also present a template toward the future engineering of catalysts to generate uniquely bioactive mitomycins.
Assuntos
Metiltransferases/química , Mitomicina/química , S-Adenosil-Homocisteína/química , Sequência de Aminoácidos , Proteínas de Bactérias , Sítios de Ligação , Cristalografia por Raios X , Metiltransferases/metabolismo , Mitomicina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , S-Adenosil-Homocisteína/metabolismo , Alinhamento de Sequência , Streptomyces/enzimologia , Homologia Estrutural de ProteínaRESUMO
The enediyne antibiotic calicheamicin (CLM) gamma(1)(I) is a prominent antitumor agent that is targeted to DNA by a novel aryltetrasaccharide comprised of an aromatic unit and four unusual carbohydrates. Herein we report the heterologous expression and the biochemical characterization of the two "internal" glycosyltransferases CalG3 and CalG2 and the structural elucidation of an enediyne glycosyltransferase (CalG3). In conjunction with the previous characterization of the "external" CLM GTs CalG1 and CalG4, this study completes the functional assignment of all four CLM GTs, extends the utility of enediyne GT-catalyzed reaction reversibility, and presents conclusive evidence of a sequential glycosylation pathway in CLM biosynthesis. This work also reveals the common GT-B structural fold can now be extended to include enediyne GTs.
Assuntos
Aminoglicosídeos/biossíntese , Aminoglicosídeos/química , Aminoglicosídeos/metabolismo , Catálise , Dimerização , Enedi-Inos/metabolismo , Glicosilação , Glicosiltransferases/química , Glicosiltransferases/isolamento & purificação , Glicosiltransferases/metabolismo , Micromonospora/enzimologia , Modelos Moleculares , Nucleotídeos/química , Nucleotídeos/metabolismo , Estrutura Quaternária de ProteínaRESUMO
The 2.65-angstroms crystal structure of the rebeccamycin 4'-O-methyltransferase RebM in complex with S-adenosyl-l-homocysteine revealed RebM to adopt a typical S-adenosylmethionine-binding fold of small molecule O-methyltransferases (O-MTases) and display a weak dimerization domain unique to MTases. Using this structure as a basis, the RebM substrate binding model implicated a predominance of nonspecific hydrophobic interactions consistent with the reported ability of RebM to methylate a wide range of indolocarbazole surrogates. This model also illuminated the three putative RebM catalytic residues (His140/141 and Asp166) subsequently found to be highly conserved among sequence-related natural product O-MTases from GC-rich bacteria. Interrogation of these residues via site-directed mutagenesis in RebM demonstrated His140 and Asp166 to be most important for catalysis. This study reveals RebM to be a member of the general acid/base-dependent O-MTases and, as the first crystal structure for a sugar O-MTase, may also present a template toward the future engineering of natural product MTases for combinatorial applications.
Assuntos
Carbazóis/metabolismo , Metiltransferases/química , Metiltransferases/genética , Carbazóis/química , Cristalização , Cristalografia por Raios X , Escherichia coli/enzimologia , Metiltransferases/isolamento & purificação , Metiltransferases/metabolismo , Modelos Moleculares , Conformação Molecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , S-Adenosil-Homocisteína/metabolismo , Estaurosporina/química , Estaurosporina/metabolismoRESUMO
The recent discovery of the first "self-sacrifice" mechanism for bacterial resistance to the enediyne antitumor antibiotics, where enediyne-induced proteolysis of the resistance protein CalC inactivates both the highly reactive metabolite and the resistance protein, revealed yet another ingenious bacterial mechanism for controlling reactive metabolites. As reported herein, the first 3D structures of CalC and CalC in complex with calicheamicin (CLM) divulge CalC to be a member of the steroidogenic acute regulatory protein (StAR)-related transfer (START) domain superfamily. In contrast to previous studies of proteins known to bind DNA-damaging natural products ( e.g ., bleomycins, mitomycins, and nine-membered chromoprotein enediynes), this is the first demonstrated involvement of a START domain fold. Consistent with the CalC self-sacrifice mechanism, CLM in complex with CalC is positioned for direct hydrogen abstraction from Gly113 to initiate the oxidative proteolysis-based resistance mechanism. These structural studies also illuminate, for the first time, a small DNA-binding region within CalC that may serve to localize CalC to the enediyne target (DNA). Given the role of START domains in nuclear/cytosolic transport and translocation, this structural study also may implicate START domains as post-endocytotic intracellular chaperones for enediyne-based therapeutics such as MyloTarg.