Covering: 2000 to 2017
Cytochrome P450 enzymes (P450s) are the most versatile biocatalysts in nature. The catalytic competence of these extraordinary hemoproteins is broadly harnessed by numerous ...chemical defenders such as bacteria, fungi, and plants for the generation of diverse and complex natural products. Rather than the common tailoring reactions (
e.g.
hydroxylation and epoxidation) mediated by the majority of biosynthetic P450s, in this review, we will focus on the unusual P450 enzymes in relation to new chemistry, skeleton construction, and structure re-shaping
via
their own unique catalytic power or the intriguing protein-protein interactions between P450s and other proteins. These uncommon P450 reactions lead to a higher level of chemical space expansion for natural products, through which a broader spectrum of bioactivities can be gained by the host organisms.
This review focuses on unusual P450 reactions related to new chemistry, skeleton construction, structure re-shaping, and protein-protein interactions in natural product biosynthesis, which play significant roles in chemical space expansion for natural products.
The superfamily of cytochrome P450 monooxygenases (P450s) is widespread in all kingdoms of life. Functionally versatile P450s are extensively involved in diverse anabolic and catabolic processes. ...P450s require electrons to be transferred by redox partners (RPs) for O2 activation and substrate monooxygenation. Unlike monotonic eukaryotic cytochrome P450 reductases, bacterial RP systems are more diverse and complicated. Recent studies have demonstrated that the type, the amount, the combination, and the mode of action of bacterial RPs can affect not only the catalytic rate and product distribution but also the type and selectivity of P450 reactions. These results are leading to a novel opinion that RPs not only function as auxiliary electron transfer proteins but are also important P450 function modulators.
Ubiquitous P450s catalyze various oxidative reactions towards an enormous number of substrates. Bacterial P450s in soluble forms represent the most diverse subset with great application value and potential.In the conventional notion, redox partners are auxiliary proteins influencing electron transfer efficiency and product distribution. Lately, growing evidence has demonstrated that redox partners can endow their paired P450s with novel functionalities and may have more unexplored physiological roles.Bacterial redox partners can form various combinations to serve different P450s in vivo. This flexibility may be important for the host microorganisms to deal with ever-changing environments.Selection and engineering of redox partners for an optimized P450–redox partner interaction interface will become an important strategy for the improvement of industrially relevant P450 catalysts.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Covering: up to 2020
As a main bioactive component of the Chinese, Indian, and American
Podophyllum
species, the herbal medicine, podophyllotoxin (PTOX) exhibits broad spectrum pharmacological ...activity, such as superior antitumor activity and against multiple viruses. PTOX derivatives (PTOXs) could arrest the cell cycle, block the transitorily generated DNA/RNA breaks, and blunt the growth-stimulation by targeting topoisomerase II, tubulin, or insulin-like growth factor 1 receptor. Since 1983, etoposide (VP-16) is being used in frontline cancer therapy against various cancer types, such as small cell lung cancer and testicular cancer. Surprisingly, VP-16 (ClinicalTrials NTC04356690) was also redeveloped to treat the cytokine storm in coronavirus disease 2019 (COVID-19) in phase II in April 2020. The treatment aims at dampening the cytokine storm and is based on etoposide in the case of central nervous system. However, the initial version of PTOX was far from perfect. Almost all podophyllotoxin derivatives, including the FDA-approved drugs VP-16 and teniposide, were seriously limited in clinical therapy due to systemic toxicity, drug resistance, and low bioavailability. To meet this challenge, scientists have devoted continuous efforts to discover new candidate drugs and have developed drug strategies. This review focuses on the current clinical treatment of PTOXs and the prospective analysis for improving druggability in the rational design of new generation PTOX-derived drugs.
Natural product podophyllotoxin exhibited superior broad spectrum antitumor and antiviral activity. Over past 30 years, scientists devoted continuous efforts to develop druggability strategies and discover new podophyllotoxin-derived drugs.
•A novel and robust Pichia pastoris yeast whole cell biocatalyst is reported.•The short chain alcohol tolerant and easily scalable biocatalyst is developed.•The biocatalyst gives improved biodiesel ...yield and productivity than Lipozyme TLIM.
A novel and robust recombinant Pichia pastoris yeast whole cell catalyst (WCC) with functional intracellular expression of Thermomyces lanuginosus lipase (Tll) was constructed and characterized for biodiesel production from waste cooking oils. This permeabilized WCC was able to convert waste cooking oils to biodiesel with 82% yield within 84h at 6% dosage whole cells. The WCC showed two fold catalytic activity of 0.73U/mg DCW compared to its commercial counterpart Lipozyme TLIM (immobilized Tll). Short chain alcohol tolerance of this WCC was significantly improved compared to Lipozyme TLIM. This beneficial property enabled it to catalyze biodiesel production efficiently with one step addition of methanol. The reusability of this biocatalyst retained 78% activity after three batch cycles. This easily prepared and cost-effective WCC showed better catalytic performance than Lipozyme TLIM with respect to biodiesel yield and productivity, thus suggesting a promising cost-effective biocatalyst for biodiesel production.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Cytochrome P450 (CYP) enzymes play crucial roles during the evolution and diversification of ancestral monocellular eukaryotes into multicellular eukaryotic organisms due to their essential ...functionalities including catalysis of housekeeping biochemical reactions, synthesis of diverse metabolites, detoxification of xenobiotics, and contribution to environmental adaptation. Eukaryotic CYPs with versatile functionalities are undeniably regarded as promising biocatalysts with great potential for biotechnological, pharmaceutical and chemical industry applications. Nevertheless, the modes of action and the challenges associated with these membrane-bound proteins have hampered the effective utilization of eukaryotic CYPs in a broader range. This review is focused on comprehensive and consolidated approaches to address the core challenges in heterologous expression of membrane-bound eukaryotic CYPs in different surrogate microbial cell factories, aiming to provide key insights for better studies and applications of diverse eukaryotic CYPs in the future. We also highlight the functional significance of the previously underrated cytochrome P450 reductases (CPRs) and provide a rational justification on the progression of CPR from auxiliary redox partner to function modulator in CYP catalysis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Mycophenolic acid (MPA) from filamentous fungi is the first natural product antibiotic to be isolated and crystallized, and a first-line immunosuppressive drug for organ transplantations and ...autoimmune diseases. However, some key biosynthetic mechanisms of such an old and important molecule have remained unclear. Here, we elucidate the MPA biosynthetic pathway that features both compartmentalized enzymatic steps and unique cooperation between biosynthetic and β-oxidation catabolism machineries based on targeted gene inactivation, feeding experiments in heterologous expression hosts, enzyme functional characterization and kinetic analysis, and microscopic observation of protein subcellular localization. Besides identification of the oxygenase MpaB′ as the long-sought key enzyme responsible for the oxidative cleavage of the farnesyl side chain, we reveal the intriguing pattern of compartmentalization for the MPA biosynthetic enzymes, including the cytosolic polyketide synthase MpaC′ and O-methyltransferase MpaG′, the Golgi apparatus-associated prenyltransferase MpaA′, the endoplasmic reticulum-bound oxygenase MpaB′ and P450-hydrolase fusion enzyme MpaDE′, and the peroxisomal acyl-coenzyme A (CoA) hydrolase MpaH′. The whole pathway is elegantly comediated by these compartmentalized enzymes, together with the peroxisomal β-oxidation machinery. Beyond characterizing the remaining outstanding steps of the MPA biosynthetic steps, our study highlights the importance of considering subcellular contexts and the broader cellular metabolism in natural product biosynthesis.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Cytochrome P450 enzymes play important roles in the biosynthesis of macrolide antibiotics by mediating a vast variety of regio- and stereoselective oxidative modifications, thus improving their ...chemical diversity, biological activities, and pharmaceutical properties. Tremendous efforts have been made on engineering the reactivity and selectivity of these useful biocatalysts. However, the 20 proteinogenic amino acids cannot always satisfy the requirement of site-directed/random mutagenesis and rational protein design of P450 enzymes. To address this issue, herein, we practice the semi-rational non-canonical amino acid mutagenesis for the pikromycin biosynthetic P450 enzyme PikC, which recognizes its native macrolide substrates with a 12- or 14-membered ring macrolactone linked to a deoxyamino sugar through a unique sugar-anchoring mechanism. Based on a semi-rationally designed substrate binding strategy, non-canonical amino acid mutagenesis at the His238 position enables the unnatural activities of several PikC mutants towards the macrolactone precursors without any sugar appendix. With the aglycone hydroxylating activities, the pikromycin biosynthetic pathway is rewired by the representative mutant PikC
carrying a p-acetylphenylalanine residue at the His238 position and a promiscuous glycosyltransferase. Moreover, structural analysis of substrate-free and three different enzyme-substrate complexes of PikC
provides significant mechanistic insights into the substrate binding and catalytic selectivity of this paradigm biosynthetic P450 enzyme.
Plants synthesize metabolites to adapt to a continuously changing environment. Metabolite biosynthesis often occurs in response to the tissue-specific combinatorial developmental cues that are ...transcriptionally regulated. Polyphyllins are the major bioactive components in Paris species that demonstrate hemostatic, anti-inflammatory and antitumor effects and have considerable market demands. However, the mechanisms underlying polyphyllin biosynthesis and regulation during plant development have not been fully elucidated. Tissue samples of P. polyphylla var. yunnanensis during the four dominant developmental stages were collected and investigated using high-performance liquid chromatography and RNA sequencing. Polyphyllin concentrations in the different tissues were found to be highly dynamic across developmental stages. Specifically, decreasing trends in polyphyllin concentration were observed in the aerial vegetative tissues, whereas an increasing trend was observed in the rhizomes. Consistent with the aforementioned polyphyllin concentration trends, different patterns of spatiotemporal gene expression in the vegetative tissues were found to be closely related with polyphyllin biosynthesis. Additionally, molecular dissection of the pathway components revealed 137 candidate genes involved in the upstream pathway of polyphyllin backbone biosynthesis. Furthermore, gene co-expression network analysis revealed 74 transcription factor genes and one transporter gene associated with polyphyllin biosynthesis and allocation. Our findings outline the framework for understanding the biosynthesis and accumulation of polyphyllins during plant development and contribute to future research in elucidating the molecular mechanism underlying polyphyllin regulation and accumulation in P. polyphylla.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Rifamycin-derived drugs, including rifampin, rifabutin, rifapentine, and rifaximin, have long been used as first-line therapies for the treatment of tuberculosis and other deadly infections. However, ...the late steps leading to the biosynthesis of the industrially important rifamycin SV and B remain largely unknown. Here, we characterize a network of reactions underlying the biosynthesis of rifamycin SV, S, L, O, and B. The two-subunit transketolase Rif15 and the cytochrome P450 enzyme Rif16 are found to mediate, respectively, a unique C-O bond formation in rifamycin L and an atypical P450 ester-to-ether transformation from rifamycin L to B. Both reactions showcase interesting chemistries for these two widespread and well-studied enzyme families.
Global navigation satellite system (GNSS) plays a crucial role in many fields, such as aerospace and transportation. Integrity is the measure of trust used in GNSS positioning especially in ...safety-critical applications. Advanced receiver autonomous integrity monitoring (ARAIM), taking full advantage of multi-constellation GNSS, shows huge potential to provide vertical navigation in civil aviation en route navigation and terminal approaches. However, the multi-constellation ARAIM also greatly exposes computational complexity and potential performance hazards in fault modes determination and fault-tolerant positioning. From the perspective of integrity risk control, rather than the pursuit of better positioning accuracy blindly for safety-critical applications, the concept of constellation dynamic selection is proposed and implemented in ARAIM and the performance analysis is discussed in this paper. Only the best two constellations which have the best vertical geometry performance are involved in ARAIM calculation anytime anywhere. The proposed method shows superiority in both integrity availability and computational complexity in both simulations and actual GNSS signal experiments. While the computational complexity is less than 10% of that using four constellations, 100% availability under LPV-200 criteria can be achieved in worldwide coverage experiment. The proposed method also overcomes the shortcomings of ARAIM with two fixed constellations and shows good robustness under depleted scenarios. Furthermore, the statistics results from observation stations proved the applicability and generality of the proposed method under current developing GNSS constellations.
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