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A facile and efficient protocol for P(O)-O bond formation was discovered through NaIO4/air-initiated phosphorylation of alcohols with H-phosphine oxides in water. This reaction showed good functional group tolerance and a broad substrate scope, providing an alternative method for constructing P(O)-O bonds. Mechanistic studies suggested that a phosphoryl radical-involving process from H-phosphine oxides facilitated the phosphorylation of alcohols under air.
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Ruthenium (Ru) nanoparticles dispersed on carbon support are promising electrocatalysts for hydrogen evolution reaction (HER) due to strong electronic metal-carbon interactions (EMCIs). Defects engineering in carbon supports is an effective strategy to adjust EMCIs. We prepared nitrogen/sulfur co-doped carbon supported Ru nanoparticles (Ru@N/S-LC) using sodium lignosulfonate and urea as feedstocks. Intrinsic S dopants from sodium lignosulfonate create rich S defects, thus enhancing the EMCIs within Ru@N/S-LC, leading a faster electron transfer between Ru nanoparticles and N/S-LC compared with N-doped carbon supported Ru nanoparticles (Ru@N-CC). The resulting Ru@N/S-LC exhibits an enhanced work function and a down-shifted d-band center, inducing stronger electron capturing ability and weaker hydrogen desorption energy than Ru@N-CC. Ru@N/S-LC requires only 7 and 94 mV overpotential in acidic medium and alkaline medium to achieve a current density of 10 mA cm-2. Density Functional Theory (DFT) calculations were utilized to clarify the impact of sulfur (S) doping and the mechanism underlying the notable catalytic activity of Ru@N/S-LC. This study offers a perspective for utilizing the natural dopants of biomass to adjust the EMCIs for electrocatalysts.
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Cytochrome P450 enzymes are promising biocatalysts for industrial use because they catalyze site-selective C-H oxidation and have diverse catalytic reactions and a broad substrate range. In this study, the 2α-hydroxylation activity of CYP154C2 from Streptomyces avermitilis MA-4680T toward androstenedione (ASD) was identified by an in vitro conversion assay. The testosterone (TES)-bound structure of CYP154C2 was solved at 1.42 Å, and this structure was used to design eight mutants, including single, double, and triple mutants, to improve the conversion efficiency. Mutants L88F/M191F and M191F/V285L were found to enhance the conversion rates significantly (i.e., 8.9-fold and 7.4-fold for TES, 46.5-fold and 19.5-fold for ASD, respectively) compared with the wild-type (WT) enzyme while retaining high 2α-position selectivity. The substrate binding affinity of the L88F/M191F mutant toward TES and ASD was enhanced compared with that of WT CYP154C2, supporting the measured increase in the conversion efficiencies. Moreover, the total turnover number and kcat/Km of the L88F/M191F and M191F/V285L mutants increased significantly. Interestingly, all mutants containing L88F generated 16α-hydroxylation products, suggesting that L88 in CYP154C2 plays a vital role in substrate selectivity and that the amino acid corresponding to L88 in the 154C subfamily affects the orientation of steroid binding and substrate selectivity. IMPORTANCE Hydroxylated derivatives of steroids play essential roles in medicine. Cytochrome P450 enzymes selectively hydroxylate methyne groups on steroids, which can dramatically change their polarity, biological activity and toxicity. There is a paucity of reports on the 2α-hydroxylation of steroids, and documented 2α-hydroxylate P450s show extremely low conversion efficiency and/or low regio- and stereoselectivity. This study conducted crystal structure analysis and structure-guided rational engineering of CYP154C2 and efficiently enhanced the conversion efficiency of TES and ASD with high regio- and stereoselectivity. Our results provide an effective strategy and theoretical basis for the 2α-hydroxylation of steroids, and the structure-guided rational design of P450s should facilitate P450 applications in the biosynthesis of steroid drugs.
Asunto(s)
Sistema Enzimático del Citocromo P-450 , Esteroides , Hidroxilación , Esteroides/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Oxidación-Reducción , Testosterona/metabolismo , Especificidad por SustratoRESUMEN
Due to the complexity of the synthetic process of cobalt phosphides (CoP), ongoing efforts concentrate on simplifying the preparation process of CoP. In this work, amino tris(methylene phosphonic acid) (ATMP, L1) and melamine (MA, L2) are assembled into two-dimensional (2D) organic nanostructures by hydrogen bonding and ionic interactions via a supramolecular assembly, which greatly weakens the coordination ability of L1 with Co2+. As the introduced L2 is rich in carbon and nitrogen, it allows the cobalt-organophosphate complex to be placed under a strongly reducing atmosphere during the high-temperature calcination process to achieve an in situ phosphating purpose. The resulting catalyst (N-CoP/NC) exhibits the sought-after enhanced oxygen reduction reaction (ORR) and hydrogen evolution reaction (HER) performance. For the ORR in 0.1 M KOH, an enhanced onset potential (0.908 V vs. RHE) and diffusion limiting current (6.280 mA cm-2) can be obtained, which is comparable to those of 20% Pt/C (0.911 V vs. RHE, 5.380 mA cm-2). For the HER in 0.5 M H2SO4, an overpotential of 150 mV is required to drive a current of 10 mA cm-2. Moreover, Density Functional Theory (DFT) calculations reveal the catalytic pathway of N-CoP/NC.
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Cytochrome P450 enzymes (CYPs or P450s) are ubiquitous heme-dependent enzymes that catalyze the monooxygenation of non-activated C-H bonds to modify the structure of the substrate. In this study, we heterologously expressed CYP107X1 from Streptomyces avermitilis and conducted inâ vitro substrate screening using the alternative redox partners putidaredoxin and putidaredoxin reductase. CYP107X1 catalyzed the 16α-hydroxylation of progesterone with regio- and stereoselectivity. The spectroscopic analyses showed that CYP107X1 bound progesterone with a relatively high Kd value of 65.3±38.9â µM. The Km and kcat values for progesterone were estimated to be 47.7±12.0â µM and 0.30â min-1 , respectively. Furthermore, a crystal structure was obtained of CYP107X1 bound with glycerol from the buffer solution. Interestingly, a conserved threonine was replaced with asparagine in CYP107X1, indicating that it may adopt an unnatural proton transfer process and play a crucial role in its catalytic activity.
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Progesterona , Streptomyces , Sistema Enzimático del Citocromo P-450/metabolismo , Hidroxilación , Progesterona/metabolismo , Streptomyces/metabolismoRESUMEN
Reasonable control of the redox states within the catalytic units together with the interconnection degrees of the substrate is of great significance in the modulation of a well-performing transducer. Herein, a novel carbon black (CB)-modified copper metal-organic framework nanomaterial (CB@Cu-MOF) prepared at room temperature was utilized as a precursor to synthesize mixed-valent copper-oxide composite catalysts (NC/CuxO-T). By tuning the carbonization process of the precursor at different temperatures (T = 100 °C, 200 °C, 300 °C and 400 °C), the different ratio configurations of the redox-alternated CuxO portions were successfully controlled with the simultaneous effective tailoring of the defect abundance in the N-doped carbon substrate. As a result, an optimized NC/CuxO-300 electrochemical H2O2 sensor was able to present a low detection limit (0.26 µM) and decent linear ranges (0.02-1.79 mM and 2.29-9.29 mM). Our strategy using easily available initial materials with mild preparation conditions is expected to promote the practical application of the star materials in laboratories.
RESUMEN
Cytochrome P450 enzymes (P450s) are versatile biocatalysts, which insert a molecular oxygen into inactivated C-H bonds under mild conditions. CYP105D7 from Streptomyces avermitilis has been reported as a bacterial substrate-promiscuous P450 which catalyzes the hydroxylation of 1-deoxypentalenic acid, diclofenac, naringenin, compactin and steroids. In this study, CYP105D7 catalyzes hydroxylation, epoxidation and dehydrogenation of capsaicin, a pharmaceutical agent, revealing its functional diversity. The kinetic parameters of the CYP105D7 oxidation of capsaicin were determined as Km =311.60±87.30â µM and kcat =2.01±0.33â min-1 . In addition, we conducted molecular docking, mutagenesis and substrate binding analysis, indicating that Arg81 plays crucial role in the capsaicin binding and catalysis. To our best knowledge, this study presents the first report to illustrate that capsaicin can be catalyzed by prokaryotic P450s.
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Capsaicina/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Streptomyces/enzimología , Biocatálisis , Capsaicina/química , Hidrogenación , Hidroxilación , Simulación del Acoplamiento Molecular , Estructura MolecularRESUMEN
Since 1970s, aplysiatoxins (ATXs), a class of biologically active dermatoxins, were identified from the marine mollusk Stylocheilus longicauda, whilst further research indicated that ATXs were originally metabolized by cyanobacteria. So far, there have been 45 aplysiatoxin derivatives discovered from marine cyanobacteria with various geographies. Recently, we isolated two neo-debromoaplysiatoxins, neo-debromoaplysiatoxin G (1) and neo-debromoaplysiatoxin H (2) from the cyanobacterium Lyngbya sp. collected from the South China Sea. The freeze-dried cyanobacterium was extracted with liquid-liquid extraction of organic solvents, and then was subjected to multiple chromatographies to yield neo-debromoaplysiatoxin G (1) (3.6 mg) and neo-debromoaplysiatoxin H (2) (4.3 mg). They were elucidated with spectroscopic methods. Moreover, the brine shrimp toxicity of the aplysiatoxin derivatives representing differential structural classifications indicated that the debromoaplysiatoxin was the most toxic compound (half inhibitory concentration (IC50) value = 0.34 ± 0.036 µM). While neo-aplysiatoxins (neo-ATXs) did not exhibit apparent brine shrimp toxicity, but showed potent blocking action against potassium channel Kv1.5, likewise, compounds 1 and 2 with IC50 values of 1.79 ± 0.22 µM and 1.46 ± 0.14 µM, respectively. Therefore, much of the current knowledge suggests the ATXs with different structure modifications may modulate multiple cellular signaling processes in animal systems leading to the harmful effects on public health.
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Toxinas de Lyngbya/química , Toxinas de Lyngbya/toxicidad , Lyngbya , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/toxicidad , Animales , Artemia/efectos de los fármacos , Células CHO , Cricetulus , Canal de Potasio Kv1.5/antagonistas & inhibidores , Canal de Potasio Kv1.5/genética , Canal de Potasio Kv1.5/fisiologíaRESUMEN
Cytochrome P450 enzymes (P450 or CYP) are some of the most versatile biocatalysts, and offer advantages for oxidizing unreactive C-H bonds in mild conditions. In this study, we identified a novel cytochrome P450 154C2 from Streptomyces avermitilis and characterized its function in 2α-hydroxylation of testosterone with regio- and stereoselectivity. To investigate the efficiency of electron transfer, we conducted biotransformation using two different P450 redox partners-RhFRED (RhF reductase domain) from Rhodococcus sp. and Pdx (putidaredoxin)/Pdr (putidaredoxin reductase) from Pseudomonas putida and revealed that RhFRED was more effective than Pdx/Pdr, especially in vivo. The Km and kcat values for testosterone were estimated to be 0.16 ± 0.05 mM and 0.13 ± 0.02 min-1, and kcat/Km was 0.81 min-1 mM-1. We also determined the crystal structure of the substrate-free form of CYP154C2 at 1.5 Å resolution. The structure has a closed conformation, and the substrate binding pocket is narrow, which can explain the strict substrate specificity of the enzyme.
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Sistema Enzimático del Citocromo P-450/metabolismo , Streptomyces/enzimología , Testosterona/química , Testosterona/metabolismo , Sitios de Unión , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Glucosafosfato Deshidrogenasa/metabolismo , Hidroxilación , Cinética , Modelos Moleculares , NADH NADPH Oxidorreductasas/metabolismo , Estereoisomerismo , Especificidad por SustratoRESUMEN
Our previous study showed that CYP105D7, a substrate-promiscuous P450, catalyzes the hydroxylation of 1-deoxypentalenic acid, diclofenac, naringenin, and compactin. In this study, 14 steroid compounds were screened using recombinant Escherichia coli cells harboring genes encoding CYP105D7 and redox partners (Pdx/Pdr, RhFRED, and FdxH/FprD), and the screening identified steroid A-ring 2ß- and D-ring 16ß-hydroxylation activity. Wild-type CYP105D7 was able to catalyze the hydroxylation of five steroids (testosterone, progesterone, 4-androstene-3,17-dione, adrenosterone, and cortisone) with low (<10%) conversion rates. Structure-guided site-directed mutagenesis of arginine residues around the substrate entrance and active site showed that the R70A and R190A single mutants and an R70A/R190A double mutant exhibited greatly enhanced conversion rates for steroid hydroxylation. For the conversion of testosterone in particular, the R70A/R190A mutant's kcat/Km values increased 1.35-fold and the in vivo conversion rates increased significantly by almost 9-fold with high regio- and stereoselectivity. Molecular docking analysis revealed that when Arg70 and Arg190 were replaced with alanine, the volume of the substrate access and binding pocket increased 1.08-fold, which might facilitate improvement of the hydroxylation efficiency of steroids.IMPORTANCE Cytochrome P450 monooxygenases (P450s) are able to introduce oxygen atoms into nonreactive hydrocarbon compounds under mild conditions, thereby offering significant advantages compared to chemical catalysts. Promiscuous P450s with broad substrate specificity and reaction diversity have significant potential for applications in various fields, including synthetic biology. The study of the function, molecular mechanisms, and rational engineering of substrate-promiscuous P450s from microbial sources is important to fulfill this potential. Here, we present a microbial substrate-promiscuous P450, CYP105D7, which can catalyze hydroxylation of steroids. The loss of the bulky side chains of Arg70 and Arg190 in the active site and substrate entrance resulted in an up to 9-fold increase in the substrate conversion rate. These findings will support future rational and semirational engineering of P450s for applications as biocatalysts.
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Arginina/metabolismo , Escherichia coli/metabolismo , Mutagénesis Sitio-Dirigida , Esteroides/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , HidroxilaciónRESUMEN
Reliable and sensitive detection of xanthine has important medical and biological significance. In this work, a novel three-dimensional (3D) conductive polymer hydrogel of polyaniline (PAni) was feasibly prepared using aniline (Ani), amino trimethylene phosphonic acid (ATMP) and ammonium persulfate ((NH4)2S2O8) as monomer, gelatinizing agent and oxidizing agent, respectively. Protonation of aniline can be achieved by ATMP, inducing good conductivity of the obtained hydrogel. ATMP remained the chelating abilities in the conductive hydrogel, enabling further immobilization with silver nanoparticles (AgNPs) functionalized by a luminol derivative, N-(aminobutyl)-N-(ethylisoluminol) (ABEI). ABEI-Ag@PAni-ATMP exhibited an enhanced performance of solid-state electrochemiluminescence (ECL). Integrated with xanthine oxidase (XOD), the proposed biosensor can be applied in the detection of xanthine via in-situ generated hydrogen peroxide (H2O2), and present a low detection limit of 9.6â¯nM, a wide linear range (from 0.01 to 200⯵M) and excellent stability.
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Técnicas Biosensibles , Técnicas Electroquímicas , Xantina/aislamiento & purificación , Sulfato de Amonio/química , Compuestos de Anilina/química , AMP Cíclico/análogos & derivados , AMP Cíclico/química , Hidrogeles/química , Peróxido de Hidrógeno/química , Límite de Detección , Luminol/análogos & derivados , Luminol/química , Nanopartículas del Metal/química , Polímeros/química , Xantina/química , Xantina Oxidasa/químicaRESUMEN
Bacterial secondary metabolites have huge application potential in multiple industries. Biosynthesis of bacterial secondary metabolites are commonly encoded in a set of genes that are organized in the secondary metabolism biosynthetic gene clusters (SMBGCs). The development of genome sequencing technology facilitates mining bacterial SMBGCs. Marine Streptomyces is a valuable resource of bacterial secondary metabolites. In this study, 87 marine Streptomyces genomes were obtained and carried out into comparative genomic analysis, which revealed their high genetic diversity due to pan-genomes owning 123,302 orthologous clusters. Phylogenomic analysis indicated that the majority of Marine Streptomyces were classified into three clades named Clade I, II, and III, containing 23, 38, and 22 strains, respectively. Genomic annotations revealed that SMBGCs in the genomes of marine Streptomyces ranged from 16 to 84. Statistical analysis pointed out that phylotypes and ecotypes were both associated with SMBGCs distribution patterns. The Clade I and marine sediment-derived Streptomyces harbored more specific SMBGCs, which consisted of several common ones; whereas the Clade II and marine invertebrate-derived Streptomyces have more SMBGCs, acting as more plentiful resources for mining secondary metabolites. This study is beneficial for broadening our knowledge about SMBGC distribution patterns in marine Streptomyces and developing their secondary metabolites in the future.
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Organismos Acuáticos/genética , Genes Bacterianos , Familia de Multigenes , Metabolismo Secundario/genética , Streptomyces/genética , Organismos Acuáticos/metabolismo , Productos Biológicos/metabolismo , Vías Biosintéticas/genética , Genómica , Filogenia , Streptomyces/metabolismoRESUMEN
The cytochrome P450 enzymes are ubiquitous heme-thiolate proteins performing regioselective and stereoselective oxygenation reactions in cellular metabolism. Due to their broad substrate scope and catalytic versatility, P450 enzymes are also attractive candidates for many industrial and biopharmaceutical applications. For particular uses, enzyme properties of P450s can be further optimized through directed evolution, rational, and semi-rational engineering approaches, all of which introduce mutations within the P450 structures. In this review, we describe the recent applications of these P450 engineering approaches and highlight the key regions and residues that have been identified using such approaches. These "hotspots" lie within critical functional areas of the P450 structure, including the active site, the substrate access channel, and the redox partner interaction interface.
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To further increase the energy and power densities of lithium-ion batteries (LIBs), monoclinic Li3 V2 (PO4 )3 attracts much attention. However, the intrinsic low electrical conductivity (2.4 × 10-7 S cm-1 ) and sluggish kinetics become major drawbacks that keep Li3 V2 (PO4 )3 away from meeting its full potential in high rate performance. Recently, significant breakthroughs in electrochemical performance (e.g., rate capability and cycling stability) have been achieved by utilizing advanced nanotechnologies. The nanostructured Li3 V2 (PO4 )3 hybrid cathodes not only improve the electrical conductivity, but also provide high electrode/electrolyte contact interfaces, favorable electron and Li+ transport properties, and good accommodation of strain upon Li+ insertion/extraction. In this Review, light is shed on recent developments in the application of 0D (nanoparticles), 1D (nanowires and nanobelts), 2D (nanoplates and nanosheets), and 3D (nanospheres) Li3 V2 (PO4 )3 for high-performance LIBs, especially highlighting their synthetic strategies and promising electrochemical properties. Finally, the future prospects of nanostructured Li3 V2 (PO4 )3 cathodes are discussed.
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Compactin and pravastatin are competitive cholesterol biosynthesis inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase and belong to the statin drugs; however, the latter shows superior pharmacokinetic characteristics. Previously, we reported that the bacterial P450, CYP105D7, from Streptomyces avermitilis can catalyze the hydroxylation of 1-deoxypentalenic acid, diclofenac, and naringenin. Here, we demonstrate that CYP105D7 could also catalyze compactin hydroxylation in vitro. In the presence of both bacterial and cyanobacterial redox partner systems with an NADPH regeneration system, the reaction produced two hydroxylated products, including pravastatin (hydroxylated at the C6 position). The steady-state kinetic parameters were measured using the redox partners of putidaredoxin and its reductase. The Km and kcat values for compactin were 39.1 ± 8.8 µM and 1.12 ± 0.09 min-1, respectively. The kcat/Km value for compactin (0.029 min-1·µM-1) was lower than that for diclofenac (0.114 min-1·µM-1). Spectroscopic analysis showed that CYP105D7 binds to compactin with a Kd value of 17.5 ± 3.6 µM. Molecular docking analysis was performed to build a possible binding model of compactin. Comparisons of different substrates with CYP105D7 were conclusively illustrated for the first time.
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Sistema Enzimático del Citocromo P-450/metabolismo , Lovastatina/análogos & derivados , Streptomyces/metabolismo , Biotecnología/métodos , Dominio Catalítico , Sistema Enzimático del Citocromo P-450/química , Ferredoxinas/metabolismo , Hidroxilación , Cinética , Lovastatina/metabolismo , Simulación del Acoplamiento Molecular , Oxidación-Reducción , Pravastatina/química , Pravastatina/metabolismo , Espectrofotometría Ultravioleta , Streptomyces/enzimologíaRESUMEN
Diclofenac is a nonsteroidal anti-inflammatory drug. It undergoes hydroxylation by mammalian cytochrome P450 enzymes at 4'- and/or 5'-positions. A bacterial P450 enzyme, CYP105D7 from Streptomyces avermitilis, has been shown to catalyze hydroxylation of 1-deoxypentalenic acid and an isoflavone daidzein. Here, we demonstrated that CYP105D7 also catalyzes hydroxylation of diclofenac at the C4'-position. A spectroscopic analysis showed that CYP105D7 binds diclofenac in a slightly cooperative manner with an affinity of 65 µM and a Hill coefficient of 1.16. The crystal structure of CYP105D7 in complex with diclofenac was determined at 2.2 Å resolution. The distal pocket of CYP105D7 contains two diclofenac molecules, illustrating drug recognition with a double-ligand-binding mode. The C3' and C4' atoms of the dichlorophenyl ring of one diclofenac molecule are positioned near the heme iron, suggesting that it is positioned appropriately for aromatic hydroxylation to yield the 4'-hydroxylated product. However, recognition of diclofenac by CYP105D7 was completely different from that of rabbit CYP2C5, which binds one diclofenac molecule with a cluster of water molecules. The distal pocket of CYP105D7 contains four arginine residues, forming a wall of the substrate-binding pocket, and the arginine residues are conserved in bacterial P450s in the CYP105 family.
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Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Diclofenaco/metabolismo , Arginina , Sitios de Unión , Cristalografía por Rayos X , Sistema Enzimático del Citocromo P-450/genética , Diclofenaco/química , Hidroxilación , Modelos Moleculares , Conformación Proteica , Streptomyces/enzimologíaRESUMEN
Pentalenic acid (1) has been isolated from many Streptomyces sp. as a co-metabolite of the sesquiterpenoid antibiotic pentalenolactone and related natural products. We have previously reported the identification of a 13.4-kb gene cluster in the genome of Streptomyces avermitilis implicated in the biosynthesis of the pentalenolactone family of metabolites consisting of 13 open reading frames. Detailed molecular genetic and biochemical studies have revealed that at least seven genes are involved in the biosynthesis of the newly discovered metabolites, neopentalenoketolactone, but no gene specifically dedicated to the formation of pentalenic acid (1) was evident in the same gene cluster. The wild-type strain of S. avermitilis, as well as its derivatives, mainly produce pentalenic acid (1), together with neopentalenoketolactone (9). Disruption of the sav7469 gene encoding a cytochrome P450 (CYP105D7), members of which class are associated with the hydroxylation of many structurally different compounds, abolished the production of pentalenic acid (1). The sav7469-deletion mutant derived from SUKA11 carrying pKU462â·ptl-clusterΔptlH accumulated 1-deoxypentalenic acid (5), but not pentalenic acid (1). Reintroduction of an extra-copy of the sav7469 gene to SUKA11 Δsav7469 carrying pKU462â·ptl-clusterΔptlH restored the production of pentalenic acid (1). Recombinant CYP105D7 prepared from Escherichia coli catalyzed the oxidative conversion of 1-deoxypentalenic acid (5) to pentalenic acid (1) in the presence of the electron-transport partners, ferredoxin (Fdx) and Fdx reductase, both in vivo and in vitro. These results unambiguously demonstrate that CYP105D7 is responsible for the conversion of 1-deoxypentalenic acid (5) to pentalenic acid (1), a shunt product in the biosynthesis of the pentalenolactone family of metabolites.
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Sistema Enzimático del Citocromo P-450/metabolismo , Streptomyces/metabolismo , Escherichia coli/genética , Hidroxilación , Isoenzimas , Cinética , Familia de Multigenes , Mutagénesis Insercional , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sesquiterpenos/metabolismo , Streptomyces/enzimología , Streptomyces/genéticaRESUMEN
The polyene macrolide antibiotic filipin is widely used as a probe for cholesterol and a diagnostic tool for type C Niemann-Pick disease. Two position-specific P450 enzymes are involved in the post-polyketide modification of filipin during its biosynthesis, thereby providing molecular diversity to the "filipin complex." CYP105P1 and CYP105D6 from Streptomyces avermitilis, despite their high sequence similarities, catalyze filipin hydroxylation at different positions, C26 and C1', respectively. Here, we determined the crystal structure of the CYP105P1-filipin I complex. The distal pocket of CYP105P1 has the second largest size among P450 hydroxylases that act on macrolide substrates. Compared with previously determined substrate-free structures, the FG helices showed significant closing motion on substrate binding. The long BC loop region adopts a unique extended conformation without a B' helix. The binding site is essentially hydrophobic, but numerous water molecules are involved in recognizing the polyol side of the substrate. Therefore, the distal pocket of CYP105P1 provides a specific environment for the large filipin substrate to bind with its pro-S side of position C26 directed toward the heme iron. The ligand-free CYP105D6 structure was also determined. A small sub-pocket accommodating the long alkyl side chain of filipin I was observed in the CYP105P1 structure but was absent in the CYP105D6 structure, indicating that filipin cannot bind to CYP105D6 with a similar orientation due to steric hindrance. This observation can explain the strict regiospecificity of these enzymes.
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Proteínas Bacterianas/química , Sistema Enzimático del Citocromo P-450/química , Streptomyces/enzimología , Secuencia de Aminoácidos , Antibacterianos/química , Cristalografía por Rayos X/métodos , Filipina/química , Hemo/química , Cinética , Ligandos , Macrólidos/química , Pruebas de Sensibilidad Microbiana , Conformación Molecular , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Estereoisomerismo , Especificidad por SustratoRESUMEN
The polyene macrolide antibiotic filipin is widely used as a probe for cholesterol in biological membranes. The filipin biosynthetic pathway of Streptomyces avermitilis contains two position-specific hydroxylases, C26-specific CYP105P1 and C1'-specific CYP105D6. In this study, we describe the three X-ray crystal structures of CYP105P1: the ligand-free wild-type (WT-free), 4-phenylimidazole-bound wild-type (WT-4PI), and ligand-free H72A mutant (H72A-free) forms. The BC loop region in the WT-free structure has a unique feature; the side chain of His72 within this region is ligated to the heme iron. On the other hand, this region is highly disordered and widely open in WT-4PI and H72A-free structures, respectively. Histidine ligation of wild-type CYP105P1 was not detectable in solution, and a type II spectral change was clearly observed when 4-phenylimidazole was titrated. The H72A mutant showed spectroscopic characteristics that were almost identical to those of the wild-type protein. In the H72A-free structure, there is a large pocket that is of the same size as the filipin molecule. The highly flexible feature of the BC loop region of CYP105P1 may be required to accept a large hydrophobic substrate.