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1.
The hydroxylation activity of the Thr268Ala mutant of P450(BM3) has been shown to occur to varying degrees with small alterations in the structure of a fatty-acid substrate. Ten substrates were investigated, including straight chain, branched chain and cis-cyclopropyl substituted fatty acids with a straight-chain length that varied between 12 and 16 carbon atoms. The efficacy of the hydroxylation activity appeared to be governed by the chain length of the substrate. Substrates possessing 14 to 15 carbons afforded the highest levels of activity, which were comparable with the wild-type enzyme. Outside of this window, straight-chain fatty acids showed reduced activity over the other substrate types. These results provide a cautionary tale concerning the loss of ferryl activity in such cytochrome P450 threonine to alanine mutants, as the nature of the substrate can determine the extent to which hydroxylation chemistry is abolished. 相似文献
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Shuaiqi Meng Dr. Jia Guo Dr. Kaili Nie Prof. Tianwei Tan Haijun Xu Prof. Luo Liu 《Chembiochem : a European journal of chemical biology》2019,20(17):2232-2235
Fatty acids are versatile precursors for fuels, fine chemicals, polymers, perfumes, etc. The properties and applications of fatty acid derivatives depend on chain length and on functional groups and their positions. To tailor fatty acids for desired properties, an engineered P450 monooxygenase has been employed here for enhanced selective hydroxylation of fatty acids. After oxidation of the hydroxy groups to the corresponding ketones, Baeyer–Villiger oxidation could be applied to introduce an oxygen atom into the hydrocarbon chains to form esters, which were finally hydrolyzed to afford either hydroxylated fatty acids or dicarboxylic fatty acids. Using this strategy, we have demonstrated that the high-value-added flavors exaltolide and silvanone supra can be synthesized from stearic acid through a hydroxylation/carbonylation/esterification/hydrolysis/lactonization reaction sequence with isolated yields of about 36 % (for ω−1 hydroxylated stearic acid; 100, 60, 80, 75 % yields for the individual reactions, respectively) or 24 % (for ω−2 hydroxylated stearic acid). Ultimately, we obtained 7.91 mg of exaltolide and 13.71 mg of silvanone supra from 284.48 mg stearic acid. 相似文献
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Z Su JH Horner M Newcomb 《Chembiochem : a European journal of chemical biology》2012,13(14):2061-2064
The rates of oxidation of fatty acids by CYP119 compound I were dependent on the pH of the medium. The plot shows log?k values for reactions of acids as a function of pH, where the slopes indicate mixed third-order and fourth-order dependence on base concentration. For palmitic acid, the rate increased 50-fold over the pH range 6.8-7.3. 相似文献
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Dr. Li Zong Dr. Yan Zhang Zhengkang Shao Dr. Anita Ljubic Prof. Charlotte Jacobsen Prof. Renjun Gao Dr. Bekir Engin Eser Dr. Yingwu Wang Prof. Zheng Guo 《Chembiochem : a European journal of chemical biology》2023,24(18):e202300368
Enzymatic hydroxylation of fatty acids by Cytochrome P450s (CYPs) offers an eco-friendly route to hydroxy fatty acids (HFAs), high-value oleochemicals with various applications in materials industry and with potential as bioactive compounds. However, instability and poor regioselectivity of CYPs are their main drawbacks. A newly discovered self-sufficient CYP102 enzyme, BAMF0695 from Bacillus amyloliquefaciens DSM 7, exhibits preference for hydroxylation of sub-terminal positions (ω-1, ω-2, and ω-3) of fatty acids. Our studies show that BAMF0695 has a broad temperature optimum (over 70 % of maximal enzymatic activity retained between 20 to 50 °C) and is highly thermostable (T50 >50 °C), affording excellent adaptive compatibility for bioprocesses. We further demonstrate that BAMF0695 can utilize renewable microalgae lipid as a substrate feedstock for HFA production. Moreover, through extensive site-directed and site-saturation mutagenesis, we isolated variants with high regioselectivity, a rare property for CYPs that usually generate complex regioisomer mixtures. BAMF0695 mutants were able to generate a single HFA regiosiomer (ω-1 or ω-2) with selectivities from 75 % up to 91 %, using C12 to C18 fatty acids. Overall, our results demonstrate the potential of a recent CYP and its variants for sustainable and green production of high-value HFAs. 相似文献
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Paula Bracco Hein J. Wijma Bastian Nicolai Jhon Alexander Rodriguez Buitrago Thomas Klünemann Agustina Vila Patrick Schrepfer Wulf Blankenfeldt Dick B. Janssen Prof. Dr. Anett Schallmey 《Chembiochem : a European journal of chemical biology》2021,22(6):1099-1110
CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure–function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation. 相似文献
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Xiangquan Qin Yiping Jiang Jie Chen Fuquan Yao Panxia Zhao Longyi Jin Zhiqi Cong 《International journal of molecular sciences》2022,23(14)
We recently developed an artificial P450–H2O2 system assisted by dual-functional small molecules (DFSMs) to modify the P450BM3 monooxygenase into its peroxygenase mode, which could be widely used for the oxidation of non-native substrates. Aiming to further improve the DFSM-facilitated P450–H2O2 system, a series of novel DFSMs having various unnatural amino acid groups was designed and synthesized, based on the co-crystal structure of P450BM3 and a typical DFSM, N-(ω-imidazolyl)-hexanoyl-L-phenylalanine, in this study. The size and hydrophobicity of the amino acid residue in the DFSM drastically affected the catalytic activity (up to 5-fold), stereoselectivity, and regioselectivity of the epoxidation and hydroxylation reactions. Docking simulations illustrated that the differential catalytic ability among the DFSMs is closely related to the binding affinity and the distance between the catalytic group and heme iron. This study not only enriches the DFSM toolbox to provide more options for utilizing the peroxide-shunt pathway of cytochrome P450BM3, but also sheds light on the great potential of the DFSM-driven P450 peroxygenase system in catalytic applications based on DFSM tunability. 相似文献
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Budde M Morr M Schmid RD Urlacher VB 《Chembiochem : a European journal of chemical biology》2006,7(5):789-794
Highly branched fatty acids, the main components of the preen-gland waxes of the domestic goose and the Muscovy duck, and their derivatives are promising chiral precursors for the synthesis of macrolide antibiotics. The key step in the utilisation of these compounds is their regioselective hydroxylation, which cannot be achieved in a classical chemical approach. Three P450 monooxygenases, CYP102A1, CYP102A2 and CYP102A3, demonstrating high turnover numbers in the hydroxylation of iso and anteiso fatty acids (>400 min(-1)), were tested for their activity towards these substrates. CYP102A1 from Bacillus megaterium and its A74G F87V L188Q triple mutant hydroxylate a variety of these substrates with high activity and regioselectivity. In all cases, the triple mutant showed much higher activities than the wild-type enzyme. The binding constants, determined for wild-type CYP102A1 and the triple mutant with tetramethylnonanol as substrate, were >200 microM and approximately 23 microM, respectively. Data derived from binding analysis support the differences in activity found for the wild-type CYP102A1 and the triple mutant. Surprisingly, CYP102A2 and CYP102A3 from Bacillus subtilis did not show any activity. Substrate binding spectra, recorded to investigate substrate accessibility to the enzyme's active sites, revealed that the substrates either could not access the active site of the Bacillus subtilis monooxygenases, or did not come into proximity with the heme. 相似文献
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Peter Meinhold Matthew&#x;W. Peters Adam Hartwick Alisha&#x;R. Hernandez Frances&#x;H. Arnold 《Advanced Synthesis u0026amp; Catalysis》2006,348(6):763-772
Enzymes that catalyze the terminal hydroxylation of alkanes could be used to produce more valuable chemicals from hydrocarbons. Cytochrome P450 BM3 from Bacillus megaterium hydroxylates medium‐chain fatty acids at subterminal positions at high rates. To engineer BM3 for terminal alkane hydroxylation, we performed saturation mutagenesis at selected active‐site residues of a BM3 variant that hydroxylates alkanes. Recombination of beneficial mutations generated a library of BM3 mutants that hydroxylate linear alkanes with a wide range of regioselectivities. Mutant 77‐9H exhibits 52% selectivity for the terminal position of octane. This regioselectivity is octane‐specific and does not transfer to other substrates, including shorter and longer hydrocarbons or fatty acids. These results show that BM3 can be readily molded for regioselective oxidation. 相似文献
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Cytochrome P450 reductase (CYPOR) provides electrons to all human microsomal cytochrome P450s (cyt P450s). The length and sequence of the “140s” FMN binding loop of CYPOR has been shown to be a key determinant of its redox potential and activity with cyt P450s. Shortening the “140s loop” by deleting glycine-141(ΔGly141) and by engineering a second mutant that mimics flavo-cytochrome P450 BM3 (ΔGly141/Glu142Asn) resulted in mutants that formed an unstable anionic semiquinone. In an attempt to understand the molecular basis of the inability of these mutants to support activity with cyt P450, we expressed, purified, and determined their ability to reduce ferric P450. Our results showed that the ΔGly141 mutant with a very mobile loop only reduced ~7% of cyt P450 with a rate similar to that of the wild type. On the other hand, the more stable loop in the ΔGly141/Glu142Asn mutant allowed for ~55% of the cyt P450 to be reduced ~60% faster than the wild type. Our results reveal that the poor activity of the ΔGly141 mutant is primarily accounted for by its markedly diminished ability to reduce ferric cyt P450. In contrast, the poor activity of the ΔGly141/Glu142Asn mutant is presumably a consequence of the altered structure and mobility of the “140s loop”. 相似文献
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Dr. Bekir Engin Eser Michal Poborsky Rongrong Dai Dr. Shigenobu Kishino Anita Ljubic Dr. Michiki Takeuchi Prof. Dr. Charlotte Jacobsen Prof. Dr. Jun Ogawa Prof. Dr. Peter Kristensen Prof. Dr. Zheng Guo 《Chembiochem : a European journal of chemical biology》2020,21(4):550-563
Enzymatic conversion of fatty acids (FAs) by fatty acid hydratases (FAHs) presents a green and efficient route for high-value hydroxy fatty acid (HFA) production. However, limited diversity was achieved among HFAs, to date, with respect to chain length and hydroxy position. In this study, two highly similar FAHs from Lactobacillus acidophilus were compared: FA-HY2 has a narrow substrate scope and strict regioselectivity, whereas FA-HY1 utilizes longer chain substrates and hydrates various double-bond positions. It is revealed that three active-site residues play a remarkable role in directing substrate specificity and regioselectivity of hydration. If these residues on FA-HY2 are mutated to the corresponding ones in FA-HY1, a significant expansion of substrate scope and a distinct enhancement in hydration of double bonds towards the ω-end of FAs is observed. A three-residue mutant of FA-HY2 (TM-FA-HY2) displayed an impressive reversal of regioselectivity towards linoleic acid, shifting the ratio of the HFA regioisomers (10-OH/13-OH) from 99:1 to 12:88. Notable changes in regioselectivity were also observed for arachidonic acid and for C18 polyunsaturated fatty acid substrates. In addition, TM-FA-HY2 converted eicosapentaenoic acid into its 12-hydroxy product with high conversion at the preparative scale. Furthermore, it is demonstrated that microalgae are a source of diverse FAs for HFA production. This study paves the way for tailor-made FAH design to enable the production of diverse HFAs for various applications from the polymer industry to medical fields. 相似文献
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There are two types of cytochrome P450 enzymes in nature, namely, the monooxygenases and the peroxygenases. Both enzyme classes participate in substrate biodegradation or biosynthesis reactions in nature, but the P450 monooxygenases use dioxygen, while the peroxygenases take H2O2 in their catalytic cycle instead. By contrast to the P450 monooxygenases, the P450 peroxygenases do not require an external redox partner to deliver electrons during the catalytic cycle, and also no external proton source is needed. Therefore, they are fully self-sufficient, which affords them opportunities in biotechnological applications. One specific P450 peroxygenase, namely, P450 OleTJE, reacts with long-chain linear fatty acids through oxidative decarboxylation to form hydrocarbons and, as such, has been implicated as a suitable source for the biosynthesis of biofuels. Unfortunately, the reactions were shown to produce a considerable amount of side products originating from Cα and Cβ hydroxylation and desaturation. These product distributions were found to be strongly dependent on whether the substrate had substituents on the Cα and/or Cβ atoms. To understand the bifurcation pathways of substrate activation by P450 OleTJE leading to decarboxylation, Cα hydroxylation, Cβ hydroxylation and Cα–Cβ desaturation, we performed a computational study using 3-phenylpropionate and 2-phenylbutyrate as substrates. We set up large cluster models containing the heme, the substrate and the key features of the substrate binding pocket and calculated (using density functional theory) the pathways leading to the four possible products. This work predicts that the two substrates will react with different reaction rates due to accessibility differences of the substrates to the active oxidant, and, as a consequence, these two substrates will also generate different products. This work explains how the substrate binding pocket of P450 OleTJE guides a reaction to a chemoselectivity. 相似文献
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Inside Cover: New Sesquiterpene Oxidations with CYP260A1 and CYP264B1 from Sorangium cellulosum So ce56 (ChemBioChem 18/2015) 
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下载免费PDF全文 Alexander Schifrin Martin Litzenburger Dr. Michael Ringle Dr. Thuy T. B. Ly Prof. Dr. Rita Bernhardt 《Chembiochem : a European journal of chemical biology》2015,16(18):2542-2542
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Alexander Schifrin Martin Litzenburger Dr. Michael Ringle Dr. Thuy T. B. Ly Prof. Dr. Rita Bernhardt 《Chembiochem : a European journal of chemical biology》2015,16(18):2624-2632
Sesquiterpenes are natural products derived from the common precursor farnesyl pyrophosphate (FPP) but are highly diverse in structure and function. Cytochrome P450 enzymes (P450s) exhibit the unique ability to introduce molecular oxygen into non‐activated C?H bonds. In plant biosynthetic pathways, P450s commonly derivatize sesquiterpene hydrocarbons. However, the potential of bacterial P450s for terpene derivatization is still underinvestigated. This work compares the substrate specificities and regioselectivities of the sesquiterpene hydroxylases CYP260A1 and CYP264B1 from myxobacterium Sorangium cellulosum So ce56. Four tested substrate classes (eremophilanes, humulanes, caryophyllanes, and cedranes) were converted by both P450s. The achievable variety of oxidations is demonstrated on the model substrates (+)‐nootkatone and zerumbone. Increasing the number of functionally investigated P450s, this study represents a step towards the selective derivatization of sesquiterpenes. 相似文献
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Dr. Chengchang Zhang Dr. Meiling Lu Dr. Lin Lin Prof. Dr. Zhangjian Huang Prof. Dr. Rongguang Zhang Dr. Xuri Wu Prof. Dr. Yijun Chen 《Chembiochem : a European journal of chemical biology》2020,21(16):2297-2305
Like a vast number of enzymes in nature, bacterial cytochrome P450 monooxygenases require an activated form of flavin as a cofactor for catalytic activity. Riboflavin is the precursor of FAD and FMN that serves as indispensable cofactor for flavoenzymes. In contrast to previous notions, herein we describe the identification of an electron-transfer process that is directly mediated by riboflavin for N-dealkylation by bacterial P450 monooxygenases. The electron relay from NADPH to riboflavin and then via activated oxygen to heme was proposed based on a combination of X-ray crystallography, molecular modeling and molecular dynamics simulation, site-directed mutagenesis and biochemical analysis of representative bacterial P450 monooxygenases. This study provides new insights into the electron transfer mechanism in bacterial P450 enzyme catalysis and likely in yeasts, fungi, plants and mammals. 相似文献
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Eerappa Rajakumara Dubey Saniya Priyanka Bajaj Rajanna Rajeshwari Jyotsnendu Giri Mehdi D. Davari 《International journal of molecular sciences》2023,24(1)
Cytochrome P450s are heme-containing enzymes capable of the oxidative transformation of a wide range of organic substrates. A protein scaffold that coordinates the heme iron, and the catalytic pocket residues, together, determine the reaction selectivity and regio- and stereo-selectivity of the P450 enzymes. Different substrates also affect the properties of P450s by binding to its catalytic pocket. Modulating the redox potential of the heme by substituting iron-coordinating residues changes the chemical reaction, the type of cofactor requirement, and the stereoselectivity of P450s. Around hundreds of P450s are experimentally characterized, therefore, a mechanistic understanding of the factors affecting their catalysis is increasingly vital in the age of synthetic biology and biotechnology. Engineering P450s can enable them to catalyze a variety of chemical reactions viz. oxygenation, peroxygenation, cyclopropanation, epoxidation, nitration, etc., to synthesize high-value chiral organic molecules with exceptionally high stereo- and regioselectivity and catalytic efficiency. This review will focus on recent studies of the mechanistic understandings of the modulation of heme redox potential in the engineered P450 variants, and the effect of small decoy molecules, dual function small molecules, and substrate mimetics on the type of chemical reaction and the catalytic cycle of the P450 enzymes. 相似文献