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1.
J Am Chem Soc ; 2024 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-38602511

RESUMO

Meroterpenoid clavilactones feature a unique benzo-fused ten-membered carbocyclic ring unit with an α,ß-epoxy-γ-lactone moiety, forming an intriguing 10/5/3 tricyclic nested skeleton. These compounds are good inhibitors of the tyrosine kinase, attracting a lot of chemical synthesis studies. However, the natural enzymes involved in the formation of the 10/5/3 tricyclic nested skeleton remain unexplored. Here, we identified a gene cluster responsible for the biosynthesis of clavilactone A in the basidiomycetous fungus Clitocybe clavipes. We showed that a key cytochrome P450 monooxygenase ClaR catalyzes the diradical coupling reaction between the intramolecular hydroquinone and allyl moieties to form the benzo-fused ten-membered carbocyclic ring unit, followed by the P450 ClaT that exquisitely and stereoselectively assembles the α,ß-epoxy-γ-lactone moiety in clavilactone biosynthesis. ClaR unprecedentedly acts as a macrocyclase to catalyze the oxidative cyclization of the isopentenyl to the nonterpenoid moieties to form the benzo-fused macrocycle, and a multifunctional P450 ClaT catalyzes a ten-electron oxidation to accomplish the biosynthesis of the 10/5/3 tricyclic nested skeleton in clavilactones. Our findings establish the foundation for the efficient production of clavilactones using synthetic biology approaches and provide the mechanistic insights into the macrocycle formation in the biosynthesis of fungal meroterpenoids.

2.
Appl Microbiol Biotechnol ; 108(1): 323, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38713233

RESUMO

Ergot alkaloids (EAs) are a diverse group of indole alkaloids known for their complex structures, significant pharmacological effects, and toxicity to plants. The biosynthesis of these compounds begins with chanoclavine-I aldehyde (CC aldehyde, 2), an important intermediate produced by the enzyme EasDaf or its counterpart FgaDH from chanoclavine-I (CC, 1). However, how CC aldehyde 2 is converted to chanoclavine-I acid (CC acid, 3), first isolated from Ipomoea violacea several decades ago, is still unclear. In this study, we provide in vitro biochemical evidence showing that EasDaf not only converts CC 1 to CC aldehyde 2 but also directly transforms CC 1 into CC acid 3 through two sequential oxidations. Molecular docking and site-directed mutagenesis experiments confirmed the crucial role of two amino acids, Y166 and S153, within the active site, which suggests that Y166 acts as a general base for hydride transfer, while S153 facilitates proton transfer, thereby increasing the acidity of the reaction. KEY POINTS: • EAs possess complicated skeletons and are widely used in several clinical diseases • EasDaf belongs to the short-chain dehydrogenases/reductases (SDRs) and converted CC or CC aldehyde to CC acid • The catalytic mechanism of EasDaf for dehydrogenation was analyzed by molecular docking and site mutations.


Assuntos
Aldeídos , Alcaloides de Claviceps , Aldeídos/metabolismo , Aldeídos/química , Domínio Catalítico , Alcaloides de Claviceps/biossíntese , Alcaloides de Claviceps/química , Alcaloides de Claviceps/metabolismo , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Oxirredutases/metabolismo , Oxirredutases/genética , Oxirredutases/química
3.
Metab Eng ; 69: 198-208, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34902590

RESUMO

Privileged ergot alkaloids (EAs) produced by the fungal genus Claviceps are used to treat a wide range of diseases. However, their use and research have been hampered by the challenging genetic engineering of Claviceps. Here we systematically refactored and rationally engineered the EA biosynthetic pathway in heterologous host Aspergillus nidulans by using a Fungal-Yeast-Shuttle-Vector protocol. The obtained strains allowed the production of diverse EAs and related intermediates, including prechanoclavine (PCC, 333.8 mg/L), chanoclavine (CC, 241.0 mg/L), agroclavine (AC, 78.7 mg/L), and festuclavine (FC, 99.2 mg/L), etc. This fungal platform also enabled the access to the methyl-oxidized EAs (MOEAs), including elymoclavine (EC), lysergic acid (LA), dihydroelysergol (DHLG), and dihydrolysergic acid (DHLA), by overexpressing a P450 enzyme CloA. Furthermore, by optimizing the P450 electron transfer (ET) pathway and using multi-copy of cloA, the titers of EC and DHLG have been improved by 17.3- and 9.4-fold, respectively. Beyond our demonstration of A. nidulans as a robust platform for EA overproduction, our study offers a proof of concept for engineering the eukaryotic P450s-contained biosynthetic pathways in a filamentous fungal host.


Assuntos
Claviceps , Alcaloides de Claviceps , Vias Biossintéticas/genética , Claviceps/genética , Claviceps/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Alcaloides de Claviceps/genética , Alcaloides de Claviceps/metabolismo , Saccharomyces cerevisiae/metabolismo
4.
Appl Microbiol Biotechnol ; 106(8): 2981-2991, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35389067

RESUMO

Ergot alkaloids (EAs) are among the most important bioactive natural products. FeII/α-ketoglutarate-dependent dioxygenase Aj_EasH from Aspergillus japonicus is responsible for the formation of the cyclopropyl ring of the ergot alkaloid (EA) cycloclavine (4). Herein we reconstituted the biosynthesis of 4 in vitro from prechanoclavine (1) for the first time. Additionally, an unexpected activity of asymmetric hydroxylation at the C-4 position of EA compound festuclavine (5) for Aj_EasH was revealed. Furthermore, Aj_EasH also catalyzes the hydroxylation of two more EAs 9,10-dihydrolysergol (6) and elymoclavine (7). Thus, our results proved that Aj_EasH is a promiscuous and bimodal dioxygenase that catalyzes both the formation of cyclopropyl ring in 4 and the asymmetric hydroxylation of EAs. Molecular docking (MD) revealed the substrate-binding mode as well as the catalytic mechanism of asymmetric hydroxylation, suggesting more EAs could potentially be recognized and hydroxylated by Aj_EasH. Overall, the newly discovered activity empowered Aj_EasH with great potential for producing more diverse and bioactive EA derivatives. KEY POINTS: • Aj_EasH was revealed to be a promiscuous and bimodal FeII/α-ketoglutarate-dependent dioxygenase. • Aj_EasH converted festuclavine, 9,10-dihydrolysergol, and elymoclavine to their hydroxylated derivatives. • The catalytic mechanism of Aj_EasH for hydroxylation was analyzed by molecular docking.


Assuntos
Alcaloides de Claviceps , Dioxigenase FTO Dependente de alfa-Cetoglutarato , Compostos Ferrosos , Hidroxilação , Simulação de Acoplamento Molecular
5.
Proc Natl Acad Sci U S A ; 116(12): 5499-5504, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30842286

RESUMO

Fungi have the potential to produce a large repertoire of bioactive molecules, many of which can affect the growth and development of plants. Genomic survey of sequenced biofertilizer fungi showed many secondary metabolite gene clusters are anchored by iterative polyketide synthases (IPKSs), which are multidomain enzymes noted for generating diverse small molecules. Focusing on the biofertilizer Trichoderma harzianum t-22, we identified and characterized a cryptic IPKS-containing cluster that synthesizes tricholignan A, a redox-active ortho-hydroquinone. Tricholignan A is shown to reduce Fe(III) and may play a role in promoting plant growth under iron-deficient conditions. The construction of tricholignan by a pair of collaborating IPKSs was investigated using heterologous reconstitution and biochemical studies. A regioselective methylation step is shown to be a key step in formation of the ortho-hydroquinone. The responsible methyltransferase (MT) is fused with an N-terminal pseudo-acyl carrier protein (ψACP), in which the apo state of the ACP is essential for methylation of the growing polyketide chain. The ψACP is proposed to bind to the IPKS and enable the trans MT to access the growing polyketide. Our studies show that a genome-driven approach to discovering bioactive natural products from biofertilizer fungi can lead to unique compounds and biosynthetic knowledge.


Assuntos
Arabidopsis/metabolismo , Ferro/metabolismo , Policetídeos/metabolismo , Trichoderma/genética , Arabidopsis/enzimologia , Redes e Vias Metabólicas/genética , Metilação , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Família Multigênica/genética , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Trichoderma/enzimologia , Trichoderma/metabolismo
6.
Angew Chem Int Ed Engl ; 61(24): e202201908, 2022 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-35322515

RESUMO

Although imine reductases (IREDs) are emerging as attractive reductive aminases (RedAms), their substrate scope is still narrow, and rational engineering is rare. Focusing on hydrogen bond reorganization and cavity expansion, a concise strategy combining rational cavity design, combinatorial active-site saturation test (CAST), and thermostability engineering was designed, that transformed the weakly active IR-G36 into a variant M5 with superior performance for the synthesis of (R)-3-benzylamino-1-Boc-piperidine, with a 4193-fold improvement in catalytic efficiency, a 16.2 °C improvement in Tm , and a significant increase in the e.e. value from 78 % (R) to >99 % (R). M5 exhibits broad substrate scope for the synthesis of diverse azacycloalkylamines, and the reaction was demonstrated on a hectogram-scale under industrially relevant conditions. Our study provides a compelling example of the preparation of versatile and efficient IREDs, with exciting opportunities in medicinal and process chemistry as well as synthetic biology.


Assuntos
Iminas , Oxirredutases , Aminação , Biocatálise , Iminas/química , Oxirredutases/química , Estereoisomerismo
7.
Appl Microbiol Biotechnol ; 105(16-17): 6333-6343, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34423409

RESUMO

The industrially important meta-cresol (m-cresol, 3-methylphenol) is mainly produced from fossil resources by chemical methods. The microbial production of m-cresol was rarely investigated. Herein, we constructed a platform for the overproduction of m-cresol in a modified fungus Aspergillus nidulans FGSC no. A1145∆ST∆EM, which gave a gram-level titer using starch as carbon resource. For the biosynthesis of m-cresol, the 6-methyl salicylic acid synthase (MSAS)-encoding gene patK and 6-methyl salicylic acid decarboxylase-encoding gene patG from A. clavatus were co-expressed in the host A. nidulans. Multiple strategies, including promotor engineering, gene multiplication, and fed-batch fermentation, were applied to raise the production of m-cresol, which resulted in the titers of 1.29 g/L in shaking flasks and 2.03 g/L in fed-batch culture. The chassis cell A. nidulans A1145∆ST∆EM was proved to possess better tolerance to m-cresol than yeast, as it could grow in the liquid medium containing up to 2.5 g/L of m-cresol. These results showed that A. nidulans has great potential to be further engineered for industrial production of m-cresol.Key points• m-Cresol was de novo biosynthesized by a fungal chassis cell Aspergillus nidulans.• Promoter engineering and gene multiplication implemented the fine-tuned genes expression.• The titer of m-cresol reached 2.03 g/L via fed-batch culture.


Assuntos
Aspergillus nidulans , Aspergillus nidulans/genética , Cresóis , Fermentação , Saccharomyces cerevisiae
8.
J Am Chem Soc ; 141(44): 17517-17521, 2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31621316

RESUMO

A dedicated enzyme for the formation of the central C ring in the tetracyclic ergoline of clinically important ergot alkaloids has never been found. Herein, we report a dual role catalase (EasC), unexpectedly using O2 as the oxidant, that catalyzes the oxidative cyclization of the central C ring from a 1,3-diene intermediate. Our study showcases how nature evolves the common catalase for enantioselective C-C bond construction of complex polycyclic scaffolds.


Assuntos
Catalase/química , Ergolinas/síntese química , Proteínas Fúngicas/química , Aspergillus fumigatus/enzimologia , Aspergillus nidulans/enzimologia , Ciclização , Radicais Livres/química , Modelos Químicos , Oxirredução , Saccharomyces cerevisiae/enzimologia
9.
J Am Chem Soc ; 140(22): 6991-6997, 2018 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-29741874

RESUMO

Duclauxins are dimeric and heptacyclic fungal polyketides with notable bioactivities. We characterized the cascade of redox transformations in the biosynthetic pathway of duclauxin from Talaromyces stipitatus. The redox reaction sequence is initiated by a cupin family dioxygenase DuxM that performs an oxidative cleavage of the peri-fused tricyclic phenalenone and affords a transient hemiketal-oxaphenalenone intermediate. Additional redox enzymes then morph the oxaphenoalenone into either an anhydride or a dihydrocoumarin-containing monomeric building block that is found in dimeric duxlauxins. Oxidative coupling between the monomers to form the initial C-C bond was shown to be catalyzed by a P450 monooxygenase, although the enzyme responsible for the second C-C bond formation was not found in the pathway. Collectively, the number and variety of redox enzymes used in the duclauxin pathway showcase Nature's strategy to generate structural complexity during natural product biosynthesis.


Assuntos
Dioxigenases/metabolismo , Fenalenos/metabolismo , Policetídeos/metabolismo , Talaromyces/química , Cromonas/química , Cromonas/metabolismo , Estrutura Molecular , Oxirredução , Fenalenos/química , Policetídeos/química , Talaromyces/metabolismo
10.
Nat Prod Rep ; 35(8): 792-837, 2018 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-29932179

RESUMO

Covering: up to 2018 α-Ketoglutarate (αKG, also known as 2-oxoglutarate)-dependent mononuclear non-haem iron (αKG-NHFe) enzymes catalyze a wide range of biochemical reactions, including hydroxylation, ring fragmentation, C-C bond cleavage, epimerization, desaturation, endoperoxidation and heterocycle formation. These enzymes utilize iron(ii) as the metallo-cofactor and αKG as the co-substrate. Herein, we summarize several novel αKG-NHFe enzymes involved in natural product biosyntheses discovered in recent years, including halogenation reactions, amino acid modifications and tailoring reactions in the biosynthesis of terpenes, lipids, fatty acids and phosphonates. We also conducted a survey of the currently available structures of αKG-NHFe enzymes, in which αKG binds to the metallo-centre bidentately through either a proximal- or distal-type binding mode. Future structure-function and structure-reactivity relationship investigations will provide crucial information regarding how activities in this large class of enzymes have been fine-tuned in nature.


Assuntos
Produtos Biológicos/metabolismo , Enzimas/química , Enzimas/metabolismo , Ferro/química , Ácidos Cetoglutáricos/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Carnitina/biossíntese , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Ciclopropanos/química , Ciclopropanos/metabolismo , Etilenos/biossíntese , Halogenação , Heme
12.
J Am Chem Soc ; 139(15): 5317-5320, 2017 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-28365998

RESUMO

Fungal polyketide synthases (PKSs) can function collaboratively to synthesize natural products of significant structural diversity. Here we present a new mode of collaboration between a highly reducing PKS (HRPKS) and a PKS-nonribosomal peptide synthetase (PKS-NRPS) in the synthesis of oxaleimides from the Penicillium species. The HRPKS is recruited in the synthesis of an olefin-containing free amino acid, which is activated and incorporated by the adenylation domain of the PKS-NRPS. The precisely positioned olefin from the unnatural amino acid is proposed to facilitate a scaffold rearrangement of the PKS-NRPS product to forge the maleimide and succinimide cores of oxaleimides.


Assuntos
Produtos Biológicos/metabolismo , Maleimidas/metabolismo , Penicillium/enzimologia , Policetídeo Sintases/metabolismo , Succinimidas/metabolismo , Produtos Biológicos/química , Maleimidas/química , Conformação Molecular , Policetídeo Sintases/química , Succinimidas/química
13.
J Am Chem Soc ; 139(10): 3639-3642, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28240554

RESUMO

Hydroalkoxylation is a powerful and efficient method of forming C-O bonds and cyclic ethers in synthetic chemistry. In studying the biosynthesis of the fungal natural product herqueinone, we identified an enzyme that can perform an intramolecular enantioselective hydroalkoxylation reaction. PhnH catalyzes the addition of a phenol to the terminal olefin of a reverse prenyl group to give a dihydrobenzofuran product. The enzyme accelerates the reaction by 3 × 105-fold compared to the uncatalyzed reaction. PhnH belongs to a superfamily of proteins with a domain of unknown function (DUF3237), of which no member has a previously verified function. The discovery of PhnH demonstrates that enzymes can be used to promote the enantioselective hydroalkoxylation reaction and form cyclic ethers.


Assuntos
Liases/metabolismo , Fenalenos/metabolismo , Biocatálise , Estrutura Molecular , Teoria Quântica , Estereoisomerismo
14.
Angew Chem Int Ed Engl ; 56(14): 3930-3934, 2017 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-28181382

RESUMO

Thiomarinol and mupirocin are assembled on similar polyketide/fatty acid backbones and exhibit potent antibiotic activity against methicillin-resistant Staphylococcus aureus (MRSA). They both contain a tetrasubstituted tetrahydropyran (THP) ring that is essential for biological activity. Mupirocin is a mixture of pseudomonic acids (PAs). Isolation of the novel compound mupirocin P, which contains a 7-hydroxy-6-keto-substituted THP, from a ΔmupP strain and chemical complementation experiments confirm that the first step in the conversion of PA-B into the major product PA-A is oxidation at the C6 position. In addition, nine novel thiomarinol (TM) derivatives with different oxidation patterns decorating the central THP core were isolated after gene deletion (tmlF). These metabolites are in accord with the THP ring formation and elaboration in thiomarinol following a similar order to that found in mupirocin biosynthesis, despite the lack of some of the equivalent genes. Novel mupirocin-thiomarinol hybrids were also synthesized by mutasynthesis.


Assuntos
Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Mupirocina/análogos & derivados , Mupirocina/farmacologia , Policetídeo Sintases/genética , Antibacterianos/química , Antibacterianos/metabolismo , Testes de Sensibilidade Microbiana , Conformação Molecular , Mupirocina/biossíntese , Mupirocina/química , Mutação , Policetídeo Sintases/metabolismo
15.
J Am Chem Soc ; 138(12): 4249-59, 2016 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-26978228

RESUMO

Phenalenones are polyketide natural products that display diverse structures and biological activities. The core of phenalenones is a peri-fused tricyclic ring system cyclized from a linear polyketide precursor via an unresolved mechanism. Toward understanding the unusual cyclization steps, the phn biosynthetic gene cluster responsible for herqueinone biosynthesis was identified from the genome of Penicillium herquei. A nonreducing polyketide synthase (NR-PKS) PhnA was shown to synthesize the heptaketide backbone and cyclize it into the angular, hemiketal-containing naphtho-γ-pyrone prephenalenone. The product template (PT) domain of PhnA catalyzes only the C4-C9 aldol condensation, which is unprecedented among known PT domains. The transformation of prephenalenone to phenalenone requires an FAD-dependent monooxygenase (FMO) PhnB, which catalyzes the C2 aromatic hydroxylation of prephenalenone and ring opening of the γ-pyrone ring simultaneously. Density functional theory calculations provide insights into why the hydroxylated intermediate undergoes an aldol-like phenoxide-ketone cyclization to yield the phenalenone core. This study therefore unveiled new routes and biocatalysts for polyketide cyclization.


Assuntos
Flavinas/metabolismo , Oxigenases/metabolismo , Fenalenos/química , Policetídeo Sintases/metabolismo , Catálise , Cromatografia Líquida , Ciclização , Fungos/enzimologia , Fungos/genética , Estrutura Molecular , Família Multigênica , Policetídeo Sintases/genética
16.
J Am Chem Soc ; 138(49): 15837-15840, 2016 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-27960349

RESUMO

The trans-decalin structure formed by intramolecular Diels-Alder cycloaddition is widely present among bioactive natural products isolated from fungi. We elucidated the concise three-enzyme biosynthetic pathway of the cytotoxic myceliothermophin and biochemically characterized the Diels-Alderase that catalyzes the formation of trans-decalin from an acyclic substrate. Computational studies of the reaction mechanism rationalize both the substrate and stereoselectivity of the enzyme.


Assuntos
Eucariotos/química , Naftalenos/metabolismo , Peptídeo Sintases/metabolismo , Policetídeo Sintases/metabolismo , Biocatálise , Reação de Cicloadição , Eucariotos/metabolismo , Euryarchaeota/enzimologia , Naftalenos/química , Peptídeo Sintases/química , Policetídeo Sintases/química
17.
J Nat Prod ; 79(8): 2066-74, 2016 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-27462726

RESUMO

Genome mining of the fungus Mucor irregularis (formerly known as Rhizomucor variabilis) revealed the presence of various gene clusters for secondary metabolite biosynthesis, including several terpene-based clusters. Investigation into the chemical diversity of M. irregularis QEN-189, an endophytic fungus isolated from the fresh inner tissue of the marine mangrove plant Rhizophora stylosa, resulted in the discovery of 20 structurally diverse indole-diterpenes including six new compounds, namely, rhizovarins A-F (1-6). Among them, compounds 1-3 represent the most complex members of the reported indole-diterpenes. The presence of an unusual acetal linked to a hemiketal (1) or a ketal (2 and 3) in an unprecedented 4,6,6,8,5,6,6,6,6-fused indole-diterpene ring system makes them chemically unique. Their structures and absolute configurations were elucidated by spectroscopic analysis, modified Mosher's method, and chemical calculations. Each of the isolated compounds was evaluated for antitumor activity against HL-60 and A-549 cell lines.


Assuntos
Antineoplásicos/isolamento & purificação , Diterpenos/isolamento & purificação , Medicamentos de Ervas Chinesas/isolamento & purificação , Indóis/isolamento & purificação , Mucor/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Cristalografia por Raios X , Diterpenos/química , Diterpenos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/farmacologia , Células HL-60 , Humanos , Indóis/química , Indóis/farmacologia , Estrutura Molecular , Rhizophoraceae/microbiologia
18.
J Am Chem Soc ; 136(14): 5501-7, 2014 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-24625190

RESUMO

Mupirocin, a clinically important antibiotic produced via a trans-AT Type I polyketide synthase (PKS) in Pseudomonas fluorescens, consists of a mixture of mainly pseudomonic acids A, B, and C. Detailed metabolic profiling of mutant strains produced by systematic inactivation of PKS and tailoring genes, along with re-feeding of isolated metabolites to mutant stains, has allowed the isolation of a large number of novel metabolites, identification of the 10,11-epoxidase, and full characterization of the mupirocin biosynthetic pathway, which proceeds via major (10,11-epoxide) and minor (10,11-alkene) parallel pathways.


Assuntos
Mupirocina/biossíntese , Policetídeo Sintases/metabolismo , Pseudomonas fluorescens/enzimologia , Conformação Molecular , Mupirocina/química , Policetídeo Sintases/genética , Pseudomonas fluorescens/metabolismo
19.
Org Lett ; 26(36): 7565-7570, 2024 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-39230034

RESUMO

Less steric ketones exhibited low stereoselectivity toward M5 due to their difficulty in restricting the free rotation of the imine intermediate. An engineered enantio-complementary imine reductase from M5 was obtained with catalytic activity. We identified four key residues that play essential roles in controlling stereoselectivity. Two mutants, I149Y-W234L (up to 99%S ee) and L200M-F260M (up to 99%R ee), were achieved, showing excellent stereoselectivity toward the tested substrates, offering valuable biocatalysts for synthesizing alkylated amphetamines.


Assuntos
Anfetaminas , Iminas , Oxirredutases , Estrutura Molecular , Estereoisomerismo , Iminas/química , Oxirredutases/metabolismo , Oxirredutases/química , Anfetaminas/química , Anfetaminas/síntese química , Alquilação , Catálise , Biocatálise
20.
Nat Commun ; 15(1): 7682, 2024 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-39227380

RESUMO

The inversion of substrate size specificity is an evolutionary roadblock for proteins. The Duf4243 dioxygenases GedK and BTG13 are known to catalyze the aromatic cleavage of bulky tricyclic hydroquinone. In this study, we discover a Duf4243 dioxygenase PaD that favors small monocyclic hydroquinones from the penicillic-acid biosynthetic pathway. Sequence alignments between PaD and GedK and BTG13 suggest PaD has three additional motifs, namely motifs 1-3, distributed at different positions in the protein sequence. X-ray crystal structures of PaD with the substrate at high resolution show motifs 1-3 determine three loops (loops 1-3). Most intriguing, loops 1-3 stack together at the top of the pocket, creating a lid-like tertiary structure with a narrow channel and a clearly constricted opening. This drastically changes the substrate specificity by determining the entry and binding of much smaller substrates. Further genome mining suggests Duf4243 dioxygenases with motifs 1-3 belong to an evolutionary branch that is extensively involved in the biosynthesis of natural products and has the ability to degrade diverse monocyclic hydroquinone pollutants. This study showcases how natural enzymes alter the substrate specificity fundamentally by incorporating new small motifs, with a fixed overall scaffold-architecture. It will also offer a theoretical foundation for the engineering of substrate specificity in enzymes and act as a guide for the identification of aromatic dioxygenases with distinct substrate specificities.


Assuntos
Motivos de Aminoácidos , Dioxigenases , Especificidade por Substrato , Dioxigenases/metabolismo , Dioxigenases/genética , Dioxigenases/química , Cristalografia por Raios X , Hidroquinonas/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Sequência de Aminoácidos , Modelos Moleculares , Alinhamento de Sequência
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