Structure-guided engineering of a flavin-containing monooxygenase for the efficient production of indirubin
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ABSTRACT:
SUBMITTER: Sun B
PROVIDER: S-EPMC10991670 | biostudies-literature | 2022 Jun
REPOSITORIES: biostudies-literature
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Project description:Indigo is currently produced by a century-old petrochemical-based process, therefore it is highly attractive to develop a more environmentally benign and efficient biotechnological process to produce this timeless dye. Flavin-containing monooxygenases (FMOs) are able to oxidize a wide variety of substrates. In this paper we show that the bacterial mFMO can be adapted to improve its ability to convert indole into indigo. The improvement was achieved by a combination of computational and structure-inspired enzyme redesign. We showed that the thermostability and the kcat for indole could be improved 1.5-fold by screening a relatively small number of enzyme mutants. This project not only resulted in an improved biocatalyst but also provided an improved understanding of the structural elements that determine the activity of mFMO and provides hints for further improvement of the monooxygenase as biocatalyst.
| S-EPMC6940849 | biostudies-literature
Project description:Flavin-containing monooxygenases (FMOs) are one of the most important monooxygenase systems in Eukaryotes and have many important physiological functions. FMOs have also been found in bacteria; however, their physiological function is not known. Here, we report the identification and characterization of trimethylamine (TMA) monooxygenase, termed Tmm, from Methylocella silvestris, using a combination of proteomic, biochemical, and genetic approaches. This bacterial FMO contains the FMO sequence motif (FXGXXXHXXXF/Y) and typical flavin adenine dinucleotide and nicotinamide adenine dinucleotide phosphate-binding domains. The enzyme was highly expressed in TMA-grown M. silvestris and absent during growth on methanol. The gene, tmm, was expressed in Escherichia coli, and the purified recombinant protein had high Tmm activity. Mutagenesis of this gene abolished the ability of M. silvestris to grow on TMA as a sole carbon and energy source. Close homologs of tmm occur in many Alphaproteobacteria, in particular Rhodobacteraceae (marine Roseobacter clade, MRC) and the marine SAR11 clade (Pelagibacter ubique). We show that the ability of MRC to use TMA as a sole carbon and/or nitrogen source is directly linked to the presence of tmm in the genomes, and purified Tmm of MRC and SAR11 from recombinant E. coli showed Tmm activities. The tmm gene is highly abundant in the metagenomes of the Global Ocean Sampling expedition, and we estimate that 20% of the bacteria in the surface ocean contain tmm. Taken together, our results suggest that Tmm, a bacterial FMO, plays an important yet overlooked role in the global carbon and nitrogen cycles.
| S-EPMC3203794 | biostudies-literature
Project description:Elimination of nonnutritional and insoluble compounds is a critical task for any living organism. Flavin-containing monooxygenases (FMOs) attach an oxygen atom to the insoluble nucleophilic compounds to increase solubility and thereby increase excretion. Here we analyze the functional mechanism of FMO from Schizosaccharomyces pombe using the crystal structures of the wild type and protein-cofactor and protein-substrate complexes. The structure of the wild-type FMO revealed that the prosthetic group FAD is an integral part of the protein. FMO needs NADPH as a cofactor in addition to the prosthetic group for its catalytic activity. Structures of the protein-cofactor and protein-substrate complexes provide insights into mechanism of action. We propose that FMOs exist in the cell as a complex with a reduced form of the prosthetic group and NADPH cofactor, readying them to act on substrates. The 4alpha-hydroperoxyflavin form of the prosthetic group represents a transient intermediate of the monooxygenation process. The oxygenated and reduced forms of the prosthetic group help stabilize interactions with cofactor and substrate alternately to permit continuous enzyme turnover.
| S-EPMC1502539 | biostudies-literature
Project description:Flavin-containing monooxygenases (FMOs) are, after cytochromes P450, the most important monooxygenase system in humans and are involved in xenobiotics metabolism and variability in drug response. The x-ray structure of a soluble prokaryotic FMO from Methylophaga sp. strain SK1 has been solved at 2.6-A resolution and is now the protein of known structure with the highest sequence similarity to human FMOs. The structure possesses a two-domain architecture, with both FAD and NADP(+) well defined by the electron density maps. Biochemical analysis shows that the prokaryotic enzyme shares many functional properties with mammalian FMOs, including substrate specificity and the ability to stabilize the hydroperoxyflavin intermediate that is crucial in substrate oxygenation. On the basis of their location in the structure, the nicotinamide ring and the adjacent ribose of NADP(+) turn out to be an integral part of the catalytic site being actively engaged in the stabilization of the oxygenating intermediate. This feature suggests that NADP(H) has a moonlighting role, in that it adopts two binding modes that allow it to function in both flavin reduction and oxygen reactivity modulation, respectively. We hypothesize that a relative domain rotation is needed to bring NADP(H) to these distinct positions inside the active site. Localization of mutations in human FMO3 that are known to cause trimethylaminuria (fish-odor syndrome) in the elucidated FMO structure provides a structural explanation for their biological effects.
| S-EPMC2373336 | biostudies-literature
Project description:Esters are valuable aroma compounds and can be produced enzymatically by Baeyer-Villiger monooxygenases (BVMOs) from (aliphatic) ketone precursors. However, a genetically encoded biosensor system for the assessment of BVMO activity and the detection of reaction products is missing. In this work, we assembled a synthetic enzyme cascade - featuring an esterase, an alcohol dehydrogenase, and LuxAB - in the heterologous host Escherichia coli. Target esters are produced by a BVMO, subsequently cleaved, and the corresponding alcohol oxidized through the artificial pathway. Ultimately, aldehyde products are detected in vivo by LuxAB, a luciferase from Photorhabdus luminescens that emits bioluminescence upon the oxidation of aldehydes to the corresponding carboxylates. This biosensor system greatly accelerated the screening and selection of active BVMO variants from a focused library, omitting commonly used low-throughput chromatographic analysis. Engineered enzymes accepted linear aliphatic ketones such as 2-undecanone and 2-dodecanone and exhibited improved ester formation.
| S-EPMC11727011 | biostudies-literature
Project description:Owing to zero-calorie, high-intensity sweetness and good taste profile, the plant-derived sweetener rebaudioside D (Reb D) has attracted great interest to replace sugars. However, low content of Reb D in stevia rebaudiana Bertoni as well as low soluble expression and enzymatic activity of plant-derived glycosyltransferase in Reb D preparation restrict its commercial usage. To address these problems, a novel glycosyltransferase YojK from Bacillus subtilis 168 with the ability to glycosylate Reb A to produce Reb D was identified. Then, structure-guided engineering was performed after solving its crystal structure. A variant YojK-I241T/G327N with 7.35-fold increase of the catalytic activity was obtained, which allowed to produce Reb D on a scale preparation with a great yield of 91.29%. Moreover, based on the results from molecular docking and molecular dynamics simulations, the improvement of enzymatic activity of YojK-I241T/G327N was ascribed to the formation of new hydrogen bonds between the enzyme and substrate or uridine diphosphate glucose. Therefore, this study provides an engineered bacterial glycosyltransferase YojK-I241T/G327N with high solubility and catalytic efficiency for potential industrial scale-production of Reb D.
| S-EPMC9452701 | biostudies-literature
Project description:Busulphan (Bu) is an alkylating agent used in the conditioning regimen prior to hematopoietic stem cell transplantation (HSCT). Bu is extensively metabolized in the liver via conjugations with glutathione to form the intermediate metabolite (sulfonium ion) which subsequently is degraded to tetrahydrothiophene (THT). THT was reported to be oxidized forming THT-1-oxide that is further oxidized to sulfolane and finally 3-hydroxysulfolane. However, the underlying mechanisms for the formation of these metabolites remain poorly understood. In the present study, we performed in vitro and in vivo investigations to elucidate the involvement of flavin-containing monooxygenase-3 (FMO3) and cytochrome P450 enzymes (CYPs) in Bu metabolic pathway. Rapid clearance of THT was observed when incubated with human liver microsomes. Furthermore, among different recombinant microsomal enzymes, the highest intrinsic clearance for THT was obtained via FMO3 followed by several CYPs including 2B6, 2C8, 2C9, 2C19, 2E1 and 3A4. In Bu- or THT-treated mice, inhibition of FMO3 by phenylthiourea significantly suppressed the clearance of both Bu and THT. Moreover, the simultaneous administration of a high dose of THT (200μmol/kg) to Bu-treated mice reduced the clearance of Bu. Consistently, in patients undergoing HSCT, repeated administration of Bu resulted in a significant up-regulation of FMO3 and glutathione-S-transfrase -1 (GSTA1) genes. Finally, in a Bu-treated patient, additional treatment with voriconazole (an antimycotic drug known as an FMO3-substrate) significantly altered the Bu clearance. In conclusion, we demonstrate for the first time that FMO3 along with CYPs contribute a major part in busulphan metabolic pathway and certainly can affect its kinetics. The present results have high clinical impact. Furthermore, these findings might be important for reducing the treatment-related toxicity of Bu, through avoiding interaction with other concomitant used drugs during conditioning and hence improving the clinical outcomes of HSCT.
| S-EPMC5679629 | biostudies-literature
Project description:The objective of the study was to investigate the regulation of hepatic flavin-containing monooxygenases (Fmo) Fmo1, Fmo3, Fmo4, and Fmo5 in three different mouse models of inflammation, including treatment with Citrobacter rodentium, lipopolysaccharide (LPS), and dextran sulfate sodium (DSS). Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to evaluate the steady-state mRNA levels for the various Fmo isoforms in these mouse models of inflammation during different treatment time courses. Fmo3 mRNA was most significantly down-regulated in C. rodentium-treated female mice. Fmo1, Fmo3, and Fmo5 mRNAs were also found to be down-regulated in LPS models of inflammation. The significant down-regulation of hepatic FMO3 protein during C. rodentium treatment was confirmed with Western blot analysis of liver microsomes from treated animals. Toll-like receptor (TLR) 4 is known to be responsible for LPS signaling in association with several proteins. To investigate whether TLR4 was responsible for regulation of Fmo genes in both LPS and C. rodentium animal models, Fmo mRNA levels in female wild-type (C3H/HeOuJ) and TLR4 mutant (C3H/HeJ) mice were compared in both inflammatory models by real-time RT-PCR. The results showed that Fmo3 down-regulation during C. rodentium infection is independent of TLR4. Whereas TLR4 is likely to play only a partial role in Fmo1 gene regulation in LPS-treated animals, our results show that the down-regulation of Fmo3 and Fmo5 in this model is TLR4-dependent. Unlike cytochrome P450 regulation measured in the same mouse strains, Fmo3 expression was largely refractory to down-regulation in the DSS model of inflammatory colitis.
| S-EPMC2680514 | biostudies-literature
Project description:BackgroundCardiac fibrosis is a common pathological feature associated with adverse clinical outcome in postinjury remodeling and has no effective therapy. Using an unbiased transcriptome analysis, we identified FMO2 (flavin-containing monooxygenase 2) as a top-ranked gene dynamically expressed following myocardial infarction (MI) in hearts across different species including rodents, nonhuman primates, and human. However, the functional role of FMO2 in cardiac remodeling is largely unknown.MethodsSingle-nuclei transcriptome analysis was performed to identify FMO2 after MI; FMO2 ablation rats were generated both in genetic level using the CRISPR-cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) technology and lentivirus-mediated manner. Gain-of-function experiments were conducted using postn-promoter FMO2, miR1a/miR133a-FMO2 lentivirus, and enzymatic activity mutant FMO2 lentivirus after MI.ResultsA significant downregulation of FMO2 was consistently observed in hearts after MI in rodents, nonhuman primates, and patients. Single-nuclei transcriptome analysis showed cardiac expression of FMO2 was enriched in fibroblasts rather than myocytes. Elevated spontaneous tissue fibrosis was observed in the FMO2-null animals without external stress. In contrast, fibroblast-specific expression of FMO2 markedly reduced cardiac fibrosis following MI in rodents and nonhuman primates associated with diminished SMAD2/3 phosphorylation. Unexpectedly, the FMO2-mediated regulation in fibrosis and SMAD2/3 signaling was independent of its enzymatic activity. Rather, FMO2 was detected to interact with CYP2J3 (cytochrome p450 superfamily 2J3). Binding of FMO2 to CYP2J3 disrupted CYP2J3 interaction with SMURF2 (SMAD-specific E3 ubiquitin ligase 2) in cytosol, leading to increased cytoplasm to nuclear translocation of SMURF2 and consequent inhibition of SMAD2/3 signaling.ConclusionsLoss of FMO2 is a conserved molecular signature in postinjury hearts. FMO2 possesses a previously uncharacterized enzyme-independent antifibrosis activity via the CYP2J3-SMURF2 axis. Restoring FMO2 expression exerts potent ameliorative effect against fibrotic remodeling in postinjury hearts from rodents to nonhuman primates. Therefore, FMO2 is a potential therapeutic target for treating cardiac fibrosis following injury.
| S-EPMC9932658 | biostudies-literature
Project description:Flavin-containing monooxygenase (FMO) oxygenates drugs/xenobiotics containing a soft nucleophile through a C4a hydroperoxy-FAD intermediate. Human FMOs 1, 2 and 3, expressed in Sf9 insect microsomes, released 30-50% of O₂ consumed as H₂O₂ upon addition of NADPH. Addition of substrate had little effect on H₂O₂ production. Two common FMO2 (the major isoform in the lung) genetic polymorphisms, S195L and N413K, were examined for generation of H₂O₂. FMO2 S195L exhibited higher "leakage", producing much greater amounts of H₂O₂, than ancestral FMO2 (FMO2.1) or the N413K variant. S195L was distinct in that H₂O₂ generation was much higher in the absence of substrate. Addition of superoxide dismutase did not impact H₂O₂ release. Catalase did not reduce levels of H₂O₂ with either FMO2.1 or FMO3 but inhibited H₂O₂ generated by FMO2 allelic variants N413K and S195L. These data are consistent with FMO molecular models. S195L resides in the GxGxSG/A NADP(+) binding motif, in which serine is highly conserved (76/89 known FMOs). We hypothesize that FMO, especially allelic variants such as FMO2 S195L, may enhance the toxicity of xenobiotics such as thioureas/thiocarbamides both by generation of sulfenic and sulfinic acid metabolites and enhanced release of reactive oxygen species (ROS) in the form of H₂O₂.
| S-EPMC4116332 | biostudies-literature