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1.
J Chem Inf Model ; 61(2): 856-868, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33534558

RESUMO

Pantetheine is ubiquitous in nature in various forms of pantetheine-containing ligands (PCLs), including coenzyme A and phosphopantetheine. Lack of scalable force field libraries for PCLs has hampered the computational studies of biological macromolecules containing PCLs. We describe here the development of the first generation Pantetheine Force Field (PFF) library that is compatible with Amber force fields; parameterized using Gasteiger, AM1-BCC, or RESP charging methods combined with gaff2 and ff14SB parameter sets. In addition, a "plug-and-play" strategy was employed to enable the systematic charging of computationally expensive molecules sharing common substructural motifs. The validation studies performed on the PFF library showed promising performance where molecular dynamics (MD) simulations results were compared with experimental data of three representative systems. The PFF library represents the first force field library capable of modeling systems containing PCLs in silico and will aid in various applications including protein engineering and drug discovery.


Assuntos
Simulação de Dinâmica Molecular , Panteteína , Biblioteca Gênica , Ligantes
2.
Int J Mol Sci ; 21(20)2020 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-33066287

RESUMO

Polyketides are a large class of structurally and functionally diverse natural products with important bioactivities. Many polyketides are synthesized by reducing type II polyketide synthases (PKSs), containing transiently interacting standalone enzymes. During synthesis, ketoreductase (KR) catalyzes regiospecific carbonyl to hydroxyl reduction, determining the product outcome, yet little is known about what drives specific KR-substrate interactions. In this study, computational approaches were used to explore KR-substrate interactions based on previously solved apo and mimic cocrystal structures. We found five key factors guiding KR-substrate binding. First, two major substrate binding motifs were identified. Second, substrate length is the key determinant of substrate binding position. Third, two key residues in chain length specificity were confirmed. Fourth, phosphorylation of substrates is critical for binding. Finally, packing/hydrophobic effects primarily determine the binding stability. The molecular bases revealed here will help further engineering of type II PKSs and directed biosynthesis of new polyketides.


Assuntos
Oxirredutases do Álcool/química , Proteínas de Bactérias/química , Simulação de Acoplamento Molecular , Policetídeos/química , Oxirredutases do Álcool/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Policetídeos/metabolismo , Ligação Proteica
3.
Nat Chem ; 11(10): 913-923, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548674

RESUMO

Modular polyketide synthases and non-ribosomal peptide synthetases are molecular assembly lines that consist of several multienzyme subunits that undergo dynamic self-assembly to form a functional megacomplex. N- and C-terminal docking domains are usually responsible for mediating the interactions between subunits. Here we show that communication between two non-ribosomal peptide synthetase subunits responsible for chain release from the enacyloxin polyketide synthase, which assembles an antibiotic with promising activity against Acinetobacter baumannii, is mediated by an intrinsically disordered short linear motif and a ß-hairpin docking domain. The structures, interactions and dynamics of these subunits were characterized using several complementary biophysical techniques to provide extensive insights into binding and catalysis. Bioinformatics analyses reveal that short linear motif/ß-hairpin docking domain pairs mediate subunit interactions in numerous non-ribosomal peptide and hybrid polyketide-non-ribosomal peptide synthetases, including those responsible for assembling several important drugs. Short linear motifs and ß-hairpin docking domains from heterologous systems are shown to interact productively, highlighting the potential of such interfaces as tools for biosynthetic engineering.


Assuntos
Peptídeo Sintases/química , Polienos/química , Policetídeo Sintases/química , Cristalografia por Raios X , Simulação de Acoplamento Molecular , Peptídeo Sintases/metabolismo , Polienos/metabolismo , Policetídeo Sintases/metabolismo , Conformação Proteica
4.
Methods Enzymol ; 622: 375-409, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31155062

RESUMO

Various computational methodologies can be applied to enzymological studies on enzymes in the fatty acid, polyketide, and non-ribosomal peptide biosynthetic pathways. These multi-domain complexes are called fatty acid synthases, polyketide synthases, and non-ribosomal peptide synthetases. These mega-synthases biosynthesize chemically diverse and complex bioactive molecules, with the intermediates being chauffeured between catalytic partners via a carrier protein. Recent efforts have been made to engineer these systems to expand their product diversity. A major stumbling block is our poor understanding of the transient protein-protein and protein-substrate interactions between the carrier protein and its many catalytic partner domains and product intermediates. The innate reactivity of pathway intermediates in two major classes of polyketide synthases has frustrated our mechanistic understanding of these interactions during the biosynthesis of these natural products, ultimately impeding the engineering of these systems for the generation of engineered natural products. Computational techniques described in this chapter can aid data interpretation or used to generate testable models of these experimentally intractable transient interactions, thereby providing insight into key interactions that are difficult to capture otherwise, with the potential to expand the diversity in these systems.


Assuntos
Ácido Graxo Sintases/química , Peptídeo Sintases/química , Policetídeo Sintases/química , Animais , Bactérias/química , Bactérias/enzimologia , Produtos Biológicos/metabolismo , Ácido Graxo Sintases/metabolismo , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Peptídeo Sintases/metabolismo , Policetídeo Sintases/metabolismo , Conformação Proteica
5.
Nat Chem Biol ; 15(7): 669-671, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209348

RESUMO

Fatty acid synthases are dynamic ensembles of enzymes that can biosynthesize long hydrocarbon chains efficiently. Here we visualize the interaction between the Escherichia coli acyl carrier protein (AcpP) and ß-ketoacyl-ACP-synthase I (FabB) using X-ray crystallography, NMR, and molecular dynamics simulations. We leveraged this structural information to alter lipid profiles in vivo and provide a molecular basis for how protein-protein interactions can regulate the fatty acid profile in E. coli.


Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Proteína de Transporte de Acila/metabolismo , Proteínas de Escherichia coli/metabolismo , Ácido Graxo Sintase Tipo II/metabolismo , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/química , Proteína de Transporte de Acila/química , Cristalografia por Raios X , Escherichia coli/química , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Ácido Graxo Sintase Tipo II/química , Modelos Moleculares , Ligação Proteica
6.
Annu Rev Biochem ; 87: 503-531, 2018 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-29925265

RESUMO

Polyketides are a large family of structurally complex natural products including compounds with important bioactivities. Polyketides are biosynthesized by polyketide synthases (PKSs), multienzyme complexes derived evolutionarily from fatty acid synthases (FASs). The focus of this review is to critically compare the properties of FASs with iterative aromatic PKSs, including type II PKSs and fungal type I nonreducing PKSs whose chemical logic is distinct from that of modular PKSs. This review focuses on structural and enzymological studies that reveal both similarities and striking differences between FASs and aromatic PKSs. The potential application of FAS and aromatic PKS structures for bioengineering future drugs and biofuels is highlighted.


Assuntos
Ácido Graxo Sintases/química , Ácido Graxo Sintases/metabolismo , Policetídeo Sintases/química , Policetídeo Sintases/metabolismo , Animais , Biocatálise , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Ácido Graxo Sintases/classificação , Humanos , Modelos Moleculares , Mimetismo Molecular , Estrutura Molecular , Policetídeo Sintases/classificação , Policetídeos/química , Policetídeos/metabolismo , Domínios Proteicos , Homologia Estrutural de Proteína , Especificidade por Substrato
7.
J Am Chem Soc ; 140(15): 4961-4964, 2018 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-29620883

RESUMO

Polyketides are a large class of bioactive natural products with a wide range of structures and functions. Polyketides are biosynthesized by large, multidomain enzyme complexes termed polyketide synthases (PKSs). One of the primary challenges when studying PKSs is the high reactivity of their poly-ß-ketone substrates. This has hampered structural and mechanistic characterization of PKS-polyketide complexes, and, as a result, little is known about how PKSs position the unstable substrates for proper catalysis while displaying high levels of regio- and stereospecificity. As a first step toward a general plan to use oxetanes as carbonyl isosteres to broadly interrogate PKS chemistry, we describe the development and application of an oxetane-based PKS substrate mimic. This enabled the first structural determination of the acyl-enzyme intermediate of a ketosynthase (KS) in complex with an inert extender unit mimic. The crystal structure, in combination with molecular dynamics simulations, led to a proposed mechanism for the unique activity of DpsC, the priming ketosynthase for daunorubicin biosynthesis. The successful application of an oxetane-based polyketide mimic suggests that this novel class of probes could have wide-ranging applications to the greater biosynthetic community interested in the mechanistic enzymology of iterative PKSs.


Assuntos
Éteres Cíclicos/química , Sondas Moleculares/química , Policetídeo Sintases/química , Policetídeos/química , Sítios de Ligação , Éteres Cíclicos/metabolismo , Sondas Moleculares/metabolismo , Estrutura Molecular , Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Especificidade por Substrato
8.
ACS Chem Biol ; 13(1): 141-151, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-29161022

RESUMO

Daunorubicin is a type II polyketide, one of a large class of polyaromatic natural products with anticancer, antibiotic, and antiviral activity. Type II polyketides are formed by the assembly of malonyl-CoA building blocks, though in rare cases, biosynthesis is initiated by the incorporation of a nonmalonyl derived starter unit, which adds molecular diversity to the poly-ß-ketone backbone. Priming mechanisms for the transfer of novel starter units onto polyketide synthases (PKS) are still poorly understood. Daunorubicin biosynthesis incorporates a unique propionyl starter unit thought to be selected for by a subclass ("DpsC type") of priming ketosynthases (KS III). To date, however, no structural information exists for this subclass of KS III enzymes. Although selectivity for self-acylation with propionyl-CoA has previously been implied, we demonstrate that DpsC shows no discrimination for self-acylation or acyl-transfer to the cognate acyl carrier protein, DpsG with short acyl-CoAs. We present five crystal structures of DpsC, including apo-DpsC, acetyl-DpsC, propionyl-DpsC, butyryl-DpsC, and a cocrystal of DpsC with a nonhydrolyzable phosphopantetheine (PPant) analogue. The DpsC crystal structures reveal the architecture of the active site, the molecular determinants for catalytic activity and homology to O-malonyl transferases, but also indicate distinct differences. These results provide a structural basis for rational engineering of starter unit selection in type II polyketide synthases.


Assuntos
Daunorrubicina/metabolismo , Policetídeo Sintases/química , Policetídeo Sintases/metabolismo , Acetilcoenzima A/química , Acetilcoenzima A/metabolismo , Acilação , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Malonil Coenzima A/química , Malonil Coenzima A/metabolismo , Modelos Moleculares , Policetídeo Sintases/genética , Conformação Proteica , Streptomyces/enzimologia
9.
Angew Chem Int Ed Engl ; 56(35): 10525-10529, 2017 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-28662280

RESUMO

The ubiquitous use of π-rich five-membered heterocycles has driven the development of new methods for their synthesis for more than a century. Here, we disclose a general and reliable reaction manifold for the construction of highly substituted heterocycles through a facile Lewis-acid-catalyzed oxetane rearrangement. Notably, this methodology employs a keto-oxetane motif as a 1,4-dicarbonyl surrogate, which can be synthesized using robust alkylation or alkenylation reactions, and thus obviates the need to access 1,4-dicarbonyl compounds via umpoled starting materials. We harnessed this reactivity to generate a broad range of substituted furans and pyrroles, and extended this methodology to produce benzo-fused versions thereof.

10.
Proc Natl Acad Sci U S A ; 114(21): E4142-E4148, 2017 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-28484029

RESUMO

Product template (PT) domains from fungal nonreducing polyketide synthases (NR-PKSs) are responsible for controlling the aldol cyclizations of poly-ß-ketone intermediates assembled during the catalytic cycle. Our ability to understand the high regioselective control that PT domains exert is hindered by the inaccessibility of intrinsically unstable poly-ß-ketones for in vitro studies. We describe here the crystallographic application of "atom replacement" mimetics in which isoxazole rings linked by thioethers mimic the alternating sites of carbonyls in the poly-ß-ketone intermediates. We report the 1.8-Å cocrystal structure of the PksA PT domain from aflatoxin biosynthesis with a heptaketide mimetic tethered to a stably modified 4'-phosphopantetheine, which provides important empirical evidence for a previously proposed mechanism of PT-catalyzed cyclization. Key observations support the proposed deprotonation at C4 of the nascent polyketide by the catalytic His1345 and the role of a protein-coordinated water network to selectively activate the C9 carbonyl for nucleophilic addition. The importance of the 4'-phosphate at the distal end of the pantetheine arm is demonstrated to both facilitate delivery of the heptaketide mimetic deep into the PT active site and anchor one end of this linear array to precisely meter C4 into close proximity to the catalytic His1345. Additional structural features, docking simulations, and mutational experiments characterize protein-substrate mimic interactions, which likely play roles in orienting and stabilizing interactions during the native multistep catalytic cycle. These findings afford a view of a polyketide "atom-replaced" mimetic in a NR-PKS active site that could prove general for other PKS domains.


Assuntos
Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Biomimética , Mutagênese Sítio-Dirigida , Panteteína/isolamento & purificação , Policetídeo Sintases/química , Policetídeo Sintases/genética , Policetídeos/química , Conformação Proteica
11.
Nat Commun ; 7: 13609, 2016 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-28000660

RESUMO

Type I modular polyketide synthases assemble diverse bioactive natural products. Such multienzymes typically use malonyl and methylmalonyl-CoA building blocks for polyketide chain assembly. However, in several cases more exotic alkylmalonyl-CoA extender units are also known to be incorporated. In all examples studied to date, such unusual extender units are biosynthesized via reductive carboxylation of α, ß-unsaturated thioesters catalysed by crotonyl-CoA reductase/carboxylase (CCRC) homologues. Here we show using a chemically-synthesized deuterium-labelled mechanistic probe, and heterologous gene expression experiments that the unusual alkylmalonyl-CoA extender units incorporated into the stambomycin family of polyketide antibiotics are assembled by direct carboxylation of medium chain acyl-CoA thioesters. X-ray crystal structures of the unusual ß-subunit of the acyl-CoA carboxylase (YCC) responsible for this reaction, alone and in complex with hexanoyl-CoA, reveal the molecular basis for substrate recognition, inspiring the development of methodology for polyketide bio-orthogonal tagging via incorporation of 6-azidohexanoic acid and 8-nonynoic acid into novel stambomycin analogues.


Assuntos
Acil Coenzima A/metabolismo , Proteínas de Bactérias/metabolismo , Policetídeo Sintases/metabolismo , Policetídeos/metabolismo , Acil Coenzima A/química , Acil Coenzima A/genética , Acil-CoA Desidrogenases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Carbono-Carbono Ligases/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Estrutura Molecular , Policetídeo Sintases/química , Policetídeo Sintases/genética , Policetídeos/química , Conformação Proteica , Homologia de Sequência de Aminoácidos , Streptomyces/genética , Streptomyces/metabolismo , Especificidade por Substrato
12.
ACS Chem Biol ; 11(12): 3421-3430, 2016 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-27779377

RESUMO

Arixanthomycins are pentangular polyphenols (PP) with potent antiproliferative activities that were discovered through the heterologous expression of environmental DNA-derived gene clusters. The biosynthesis of arixanthomycin and other PPs is unusual because it requires several novel type II polyketide synthase (PKS) enzymes for its complete maturation. Most type II PKSs contain a ketoreductase (KR) that mediates the C7-C12 first ring cyclization and C-9 reduction. In contrast, based on previous studies of product analysis and genome mining, the arixanthomycin (ARX) gene cluster harbors a C-11 reducing KR (ARX 27), a C9-C14 first-ring aromatase/cyclase (ARX 19), and an unprecedented C-17 and C-19 reducing KR (ARX 21). While bioinformatics is useful for predicting novel enzymes, the functions of ARX 19, ARX 21, and ARX 27 have yet to be confirmed. Further, the structural features that predispose the ARX biosynthetic enzymes to process atypical poly-ß-ketone scaffolds remain unknown. We report the crystal structure of ARX 21, the first structure of an enzyme involved in PP biosynthesis and likely a C-17 and C-19 reducing-KR, which is structurally similar to C-15 reducing KRs. Structural comparison of ARX 21 and other C-9 reducing KRs revealed a difference in the enzyme active site that may enlighten the molecular basis of KR substrate specificity. In addition, we report the successful in vitro reconstitution of ARX 19. The structural characterization of ARX 21 in conjunction with the in vitro results of ARX 19 lays the groundwork toward a complete in vitro and structural characterization of type II PKS enzymes involved in PP biogenesis.


Assuntos
Oxirredutases do Álcool/metabolismo , Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Policetídeo Sintases/metabolismo , Polifenóis/metabolismo , Oxirredutases do Álcool/química , Sequência de Aminoácidos , Bactérias/química , Bactérias/metabolismo , Proteínas de Bactérias/química , Vias Biossintéticas , Cristalografia por Raios X , Policetídeo Sintases/química , Conformação Proteica , Multimerização Proteica
13.
Angew Chem Int Ed Engl ; 55(42): 13005-13009, 2016 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-27653519

RESUMO

In fungal non-reducing polyketide synthases (NR-PKS) the acyl-carrier protein (ACP) carries the growing polyketide intermediate through iterative rounds of elongation, cyclization and product release. This process occurs through a controlled, yet enigmatic coordination of the ACP with its partner enzymes. The transient nature of ACP interactions with these catalytic domains imposes a major obstacle for investigation of the influence of protein-protein interactions on polyketide product outcome. To further our understanding about how the ACP interacts with the product template (PT) domain that catalyzes polyketide cyclization, we developed the first mechanism-based crosslinkers for NR-PKSs. Through in vitro assays, in silico docking and bioinformatics, ACP residues involved in ACP-PT recognition were identified. We used this information to improve ACP compatibility with non-cognate PT domains, which resulted in the first gain-of-function ACP with improved interactions with its partner enzymes. This advance will aid in future combinatorial biosynthesis of new polyketides.


Assuntos
Proteína de Transporte de Acila/química , Policetídeos/química , Proteína de Transporte de Acila/metabolismo , Conformação Molecular , Simulação de Acoplamento Molecular , Policetídeos/metabolismo , Ligação Proteica , Conformação Proteica
14.
Tetrahedron ; 72(25): 3605-3608, 2016 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-27346894

RESUMO

Polyketide biosynthesis engages a series of well-timed biosynthetic operations to generate elaborate natural products from simple building blocks. Mimicry of these processes has offered practical means for total synthesis and provided a foundation for reaction discovery. We now report an unusual intramolecular trans-amidation reaction discovered while preparing stabilized probes for the study of actinorhodin biosynthesis. This rapid cyclization event offers insight into the natural cyclization process inherent to the biosynthesis of type II polyketide antibiotics.

15.
Org Lett ; 18(5): 1124-7, 2016 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-26889956

RESUMO

Many halogenases interchangeably incorporate chlorine and bromine into organic molecules. On the basis of an unsubstantiated report that the alga Ochromonas danica, a prodigious producer of chlorosulfolipids, was able to produce bromosulfolipids, we have investigated the promiscuity of its halogenases toward bromine incorporation. We have found that bromosulfolipids are produced with the exact positional and stereochemical selectivity as in the chlorosulfolipid danicalipin A when this alga is grown under modified conditions containing excess bromide ion.


Assuntos
Hidrocarbonetos Bromados/isolamento & purificação , Lipídeos/isolamento & purificação , Ochromonas/química , Hidrocarbonetos Bromados/química , Lipídeos/química , Estrutura Molecular
16.
ACS Chem Biol ; 11(4): 1137-47, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26813028

RESUMO

Cores of aromatic polyketides are essential for their biological activities. Most type II polyketide synthases (PKSs) biosynthesize these core structures involving the minimal PKS, a PKS-associated ketoreductase (KR) and aromatases/cyclases (ARO/CYCs). Oxygenases (OXYs) are rarely involved. BE-7585A is an anticancer polyketide with an angucyclic core. (13)C isotope labeling experiments suggest that its angucyclic core may arise from an oxidative rearrangement of a linear anthracyclinone. Here, we present the crystal structure and functional analysis of BexE, the oxygenase proposed to catalyze this key oxidative rearrangement step that generates the angucyclinone framework. Biochemical assays using various linear anthracyclinone model compounds combined with docking simulations narrowed down the substrate of BexE to be an immediate precursor of aklaviketone, possibly 12-deoxy-aklaviketone. The structural analysis, docking simulations, and biochemical assays provide insights into the role of BexE in BE-7585A biosynthesis and lay the groundwork for engineering such framework-modifying enzymes in type II PKSs.


Assuntos
Policetídeo Sintases/metabolismo , Tioaçúcares/metabolismo , Conformação Molecular , Oxirredução
17.
ACS Chem Biol ; 11(1): 95-103, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26473393

RESUMO

The incorporation of nonacetate starter units during type II polyketide biosynthesis helps diversify natural products. Currently, there are few enzymatic strategies for the incorporation of nonacetate starter units in type II polyketide synthase (PKS) pathways. Here we report the crystal structure of AuaEII, the anthranilate:CoA ligase responsible for the generation of anthraniloyl-CoA, which is used as a starter unit by a type II PKS in aurachin biosynthesis. We present structural and protein sequence comparisons to other aryl:CoA ligases. We also compare the AuaEII crystal structure to a model of a CoA ligase homologue, AuaE, which is present in the same gene cluster. AuaE is predicted to have the same fold as AuaEII, but instead of CoA ligation, AuaE catalyzes acyl transfer of anthranilate from anthraniloyl-CoA to the acyl carrier protein (ACP). Together, this work provides insight into the molecular basis for starter unit selection of anthranilate in type II PKS biosynthesis.


Assuntos
Modelos Moleculares , Policetídeo Sintases/química , Policetídeos/química , ortoaminobenzoatos/química , Domínio Catalítico , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Estrutura Molecular , Policetídeos/metabolismo , Homologia de Sequência , Streptomyces/enzimologia , Streptomyces/metabolismo , Especificidade por Substrato
18.
Proc Natl Acad Sci U S A ; 112(50): E6844-51, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-26631750

RESUMO

Aromatic polyketides make up a large class of natural products with diverse bioactivity. During biosynthesis, linear poly-ß-ketone intermediates are regiospecifically cyclized, yielding molecules with defined cyclization patterns that are crucial for polyketide bioactivity. The aromatase/cyclases (ARO/CYCs) are responsible for regiospecific cyclization of bacterial polyketides. The two most common cyclization patterns are C7-C12 and C9-C14 cyclizations. We have previously characterized three monodomain ARO/CYCs: ZhuI, TcmN, and WhiE. The last remaining uncharacterized class of ARO/CYCs is the di-domain ARO/CYCs, which catalyze C7-C12 cyclization and/or aromatization. Di-domain ARO/CYCs can further be separated into two subclasses: "nonreducing" ARO/CYCs, which act on nonreduced poly-ß-ketones, and "reducing" ARO/CYCs, which act on cyclized C9 reduced poly-ß-ketones. For years, the functional role of each domain in cyclization and aromatization for di-domain ARO/CYCs has remained a mystery. Here we present what is to our knowledge the first structural and functional analysis, along with an in-depth comparison, of the nonreducing (StfQ) and reducing (BexL) di-domain ARO/CYCs. This work completes the structural and functional characterization of mono- and di-domain ARO/CYCs in bacterial type II polyketide synthases and lays the groundwork for engineered biosynthesis of new bioactive polyketides.


Assuntos
Aromatase/metabolismo , Policetídeo Sintases/química , Policetídeo Sintases/metabolismo , Aromatase/química , Aromatase/genética , Modelos Moleculares , Mutagênese , Policetídeo Sintases/genética , Conformação Proteica
19.
Chem Biol ; 22(11): 1453-1460, 2015 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-26526101

RESUMO

Microbial fatty acid biosynthetic enzymes are important targets for areas as diverse as antibiotic development to biofuel production. Elucidating the molecular basis of chain length control during fatty acid biosynthesis is crucial for the understanding of regulatory processes of this fundamental metabolic pathway. In Escherichia coli, the acyl carrier protein (AcpP) plays a central role by sequestering and shuttling the growing acyl chain between fatty acid biosynthetic enzymes. FabA, a ß-hydroxyacyl-AcpP dehydratase, is an important enzyme in controlling fatty acid chain length and saturation levels. FabA-AcpP interactions are transient in nature and thus difficult to visualize. In this study, four mechanistic crosslinking probes mimicking varying acyl chain lengths were synthesized to systematically probe for modified chain length specificity of 14 FabA mutants. These studies provide evidence for the AcpP-interacting "positive patch," FabA mutations that alter substrate specificity, and the roles that the FabA "gating residues" play in chain length control.


Assuntos
Escherichia coli/enzimologia , Ácido Graxo Sintase Tipo II/metabolismo , Hidroliases/metabolismo , Proteína de Transporte de Acila/química , Proteína de Transporte de Acila/genética , Proteína de Transporte de Acila/metabolismo , Sítios de Ligação , Reagentes de Ligações Cruzadas/química , Ácido Graxo Sintase Tipo II/antagonistas & inibidores , Ácido Graxo Sintase Tipo II/genética , Hidroliases/antagonistas & inibidores , Hidroliases/genética , Simulação de Dinâmica Molecular , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Mutagênese , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Especificidade por Substrato
20.
Chem Biol ; 22(8): 1018-29, 2015 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-26235055

RESUMO

The terminal reductase (R) domain from the non-ribosomal peptide synthetase (NRPS) module MxaA in Stigmatella aurantiaca Sga15 catalyzes a non-processive four-electron reduction to produce the myxalamide family of secondary metabolites. Despite widespread use in nature, a lack of structural and mechanistic information concerning reductive release from polyketide synthase (PKS) and NRPS assembly lines principally limits our ability to redesign R domains with altered or improved activity. Here we report crystal structures for MxaA R, both in the absence and, for the first time, in the presence of the NADPH cofactor. Molecular dynamics simulations were employed to provide a deeper understanding of this domain and further identify residues critical for structural integrity, substrate binding, and catalysis. Aggregate computational and structural findings provided a basis for mechanistic investigations and, in the process, delivered a rationally altered variant with improved activity toward highly reduced substrates.


Assuntos
Álcoois/química , Peptídeo Sintases/química , Policetídeo Sintases/química , Policetídeo Sintases/metabolismo , Álcoois/síntese química , Álcoois/metabolismo , Simulação por Computador , Cristalografia por Raios X , Simulação de Dinâmica Molecular , NADP/química , NADP/metabolismo , Oxirredutases/metabolismo , Peptídeo Sintases/análise , Peptídeo Sintases/metabolismo , Polienos/química , Engenharia de Proteínas , Estrutura Terciária de Proteína , Stigmatella aurantiaca/enzimologia , Stigmatella aurantiaca/metabolismo
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