Removal of phosphoglycolate in hyperthermophilic archaea.

Many organisms that utilize the Calvin-Benson-Bassham (CBB) cycle for autotrophic growth harbor metabolic pathways to remove and/or salvage 2-phosphoglycolate, the product of the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). It has been presumed that the occurrence of 2-phosphoglycolate salvage is linked to the CBB cycle, and in particular, the C2 pathway to the CBB cycle and oxygenic photosynthesis. Here, we examined 2-phosphoglycolate salvage in the hyperthermophilic archaeon Thermococcus kodakarensis, an obligate anaerobe that harbors a Rubisco that functions in the pentose bisphosphate pathway. T. kodakarensis harbors enzymes that have the potential to convert 2-phosphoglycolate to glycine and serine, and their genes were identified by biochemical and/or genetic analyses. 2-phosphoglycolate phosphatase activity increased 1.6-fold when cells were grown under microaerobic conditions compared to anaerobic conditions. Among two candidates, TK1734 encoded a phosphatase specific for 2-phosphoglycolate, and the enzyme was responsible for 80% of the 2-phosphoglycolate phosphatase activity in T. kodakarensis cells. The TK1734 disruption strain displayed growth impairment under microaerobic conditions, which was relieved upon addition of sodium sulfide. In addition, glycolate was detected in the medium when T. kodakarensis was grown under microaerobic conditions. The results suggest that T. kodakarensis removes 2-phosphoglycolate via a phosphatase reaction followed by secretion of glycolate to the medium. As the Rubisco in T. kodakarensis functions in the pentose bisphosphate pathway and not in the CBB cycle, mechanisms to remove 2-phosphoglycolate in this archaeon emerged independent of the CBB cycle.

Direct Methane Oxidation by Copper- and Iron-Dependent Methane Monooxygenases.

Methane is a potent greenhouse gas that contributes significantly to climate change and is primarily regulated in Nature by methanotrophic bacteria, which consume methane gas as their source of energy and carbon, first by oxidizing it to methanol. The direct oxidation of methane to methanol is a chemically difficult transformation, accomplished in methanotrophs by complex methane monooxygenase (MMO) enzyme systems. These enzymes use iron or copper metallocofactors and have been the subject of detailed investigation. While the structure, function, and active site architecture of the copper-dependent particulate methane monooxygenase (pMMO) have been investigated extensively, its putative quaternary interactions, regulation, requisite cofactors, and mechanism remain enigmatic. The iron-dependent soluble methane monooxygenase (sMMO) has been characterized biochemically, structurally, spectroscopically, and, for the most part, mechanistically. Here, we review the history of MMO research, focusing on recent developments and providing an outlook for future directions of the field. Engineered biological catalysis systems and bioinspired synthetic catalysts may continue to emerge along with a deeper understanding of the molecular mechanisms of biological methane oxidation. Harnessing the power of these enzymes will necessitate combined efforts in biochemistry, structural biology, inorganic chemistry, microbiology, computational biology, and engineering.

Deciphering the diurnal rhythm regulating mechanism of flavin-containing monooxygenase 3 in mouse liver.

Circadian genes play an important role in the field of drug metabolism. Flavin-containing monooxygenase 3 is a well-known phase I enzyme which participates in metabolism of many exogenous and endogenous substances, especially production of trimethylamine N-oxide. Here, we aimed to decipher diurnal rhythms of flavin-containing monooxygenase 3 expression and activity, and explore the regulation mechanism by clock genes. Our results showed that its mRNA and protein exhibited robust diurnal rhythms in mouse liver and cell lines. Consistently, significant alterations were observed for in vitro microsomal N-oxidation rates of procainamide, which kept in line with its protein expression at different time in wild-type and reverse erythroblastosis virus α knockout mice. Further, flavin-containing monooxygenase 3 was negatively regulated by E4 promoter-binding protein 4 in AML12 and Hepa1-6 cells, while it was positively influenced by reverse erythroblastosis virus α and brain and muscle ARNT-like protein-1. Moreover, luciferase reporter assays and electrophoretic mobility shift assays showed E4 promoter-binding protein 4 inhibited the transcription of flavin-containing monooxygenase 3 by binding to a D-box1 element (-1606/-1594 bp), while brain and muscle ARNT-like protein-1 positively activated the transcription via direct binding to three E-boxes (-863/-858 bp, -507/-498 bp, and -115/-104 bp) in this enzyme promoter. Taken together, this study would be helpful to reveal the mechanism of clock-controlled drug metabolism and facilitate the practice of chrono-therapeutics.

Cleavage of natural rubber by rubber oxygenases in Gram-negative bacteria.

Bacterial degradation of natural rubber (NR) in an oxic environment is initiated by oxidative cleavage of double bonds in the NR-carbon backbone and is catalyzed by extracellular haem-containing rubber oxygenases. NR-cleavage products of sufficiently low molecular mass are taken up by the cells and metabolized for energy and biomass formation. Gram-negative and Gram-positive NR-degrading bacteria (usually) employ different types of rubber oxygenases such as RoxA and/or RoxB (most Gram-negative NR-degraders) or latex clearing protein Lcp (most Gram-positive NR-degraders). In order to find novel orthologues of Rox proteins, we have revisited databases and provide an update of Rox-like proteins. We describe the putative evolution of rubber oxygenases and confirm the presence of a third subgroup of Rox-related proteins (RoxCs), the biological function of which remains, however, unclear. We summarize the knowledge on the taxonomic position of Steroidobacter cummioxidans 35Y and related species. Comparison of genomic and biochemical features of strain 35Y with other species of the genus Steroidobacter suggests that strain 35Y represents a species of a novel genus for which the designation Aurantibaculum gen. nov. is proposed. A short summary on the capabilities of NR-degrading consortia, that could be superior in biotechnological applications compared to pure cultures, is also provided. KEY POINTS: • Three types of rubber oxygenases exist predominantly in Gram-negative microbes • S. cummioxidans 35Y contains RoxA and RoxB which are superior in activity • S. cummioxidans 35Y represents a species of a novel genus.

Heterologous Biosynthesis of the Sterol O-Acyltransferase Inhibitor Helvamide Unveils an α-Ketoglutarate-Dependent Cross-Linking Oxygenase.

We have identified the biosynthetic gene cluster (hvm) for the sterol O-acyltransferase inhibitor helvamide (1) from the genome of Aspergillus rugulosus MST-FP2007. Heterologous expression of hvm in A. nidulans produced a previously unreported analog helvamide B (5). An α-ketoglutarate-dependent oxygenase Hvm1 was shown to catalyze intramolecular cyclization of 1 to yield 5. The biosynthetic branch to the related hancockiamides and helvamides was found to be controlled by the substrate selectivity of monomodular nonribosomal peptide synthetases.

Fus-SMO: Kinetics, Biochemical Characterisation and In Silico Modelling of a Chimeric Styrene Monooxygenase Demonstrating Quantitative Coupling Efficiency.

The styrene monooxygenase, a two-component enzymatic system for styrene epoxidation, was characterised through the study of Fus-SMO - a chimera resulting from the fusion of StyA and StyB using a flexible linker. Notably, it remains debated whether the transfer of FADH2 from StyB to StyA occurs through diffusion, channeling, or a combination of both. Fus-SMO was identified as a trimer with one bound FAD molecule. In silico modelling revealed a well-distanced arrangement (45-50 Å) facilitated by the flexible linker's loopy structure. Pre-steady-state kinetics elucidated the FADox reduction intricacies (kred=110 s-1 for bound FADox), identifying free FADox binding as the rate-determining step. The aerobic oxidation of FADH2 (kox=90 s-1) and subsequent decomposition to FADox and H2O2 demonstrated StyA's protective effect on the bound hydroperoxoflavin (kdec=0.2 s-1) compared to free cofactor (kdec=1.8 s-1). At varied styrene concentrations, kox for FADH2 ranged from 80 to 120 s-1. Studies on NADH consumption vs. styrene epoxidation revealed Fus-SMO's ability to achieve quantitative coupling efficiency in solution, surpassing natural two-component SMOs. The results suggest that Fus-SMO exhibits enhanced FADH2 channelling between subunits. This work contributes to comprehending FADH2 transfer mechanisms in SMO and illustrates how protein fusion can elevate catalytic efficiency for biocatalytic applications.

Molecular and functional characterization of flavin-containing monooxygenases (FMO1-6) in tree shrews.

Flavin-containing monooxygenases (FMOs) are a family of important drug oxygenation enzymes that, in humans, consist of five functional enzymes (FMO1-5) and a pseudogene (FMO6P). The tree shrew is a non-rodent primate-like species that is used in various biomedical studies, but its usefulness in drug metabolism research has not yet been investigated. In this study, tree shrew FMO1-6 cDNAs were isolated and characterized by sequence analysis, tissue expression, and metabolic function. Compared with human FMOs, tree shrew FMOs showed sequence identities of 85-90 % and 81-89 %, respectively, for cDNA and amino acids. Phylogenetic analysis showed that each tree shrew and human FMO were closely clustered. The genomic and genetic structures of the FMO genes were conserved in tree shrews and humans. Among the five tissue types analyzed (lung, heart, kidney, small intestine, and liver), FMO3 and FMO1 mRNAs were most abundant in liver and kidney, respectively. Recombinant tree shrew FMO1-6 proteins expressed in bacterial membranes all mediated benzydamine and trimethylamine N-oxygenations and methyl p-tolyl sulfide S-oxygenation. The selective human FMO3 substrate trimethylamine was predominantly metabolized by tree shrew FMO3. Additionally, tree shrew FMO6 was active toward trimethylamine, as is cynomolgus macaque FMO6, in contrast with the absence of activity of the human FMO6P pseudogene product. Tree shrew FMO1-6, which are orthologous to human FMOs (FMO1-5 and FMO6P) were identified, and tree shrew FMO3 has functional and molecular features generally comparable to those of human FMO3 as the predominant FMO in liver.

Rare but impaired flavin-containing monooxygenase 3 (FMO3) variants reported in a recently updated Japanese mega-databank of genome resources.

Genetic variants of human flavin-containing monooxygenase 3 (FMO3) were investigated using an updated Japanese population panel containing 54,000 subjects (the previous panel contained 38,000 subjects). One stop codon mutation and six amino acid-substituted FMO3 variants were newly identified in the updated databank. Of these, two substituted variants (p.Thr329Ala and p.Arg492Trp) were previously identified in compound haplotypes with p.[(Glu158Lys; Glu308Gly)] and were associated with the metabolic disorder trimethylaminuria. Three recombinant FMO3 protein variants (p.Ser137Leu, p.Ala334Val, and p.Ile426Val) expressed in bacterial membranes had similar activities toward trimethylamine N-oxygenation (∼75-125 %) as wild-type FMO3 (117 min-1); however, the recombinant novel FMO3 variant Phe313Ile showed moderately decreased FMO3 catalytic activity (∼20 % of wild-type). Because of the known deleterious effects of FMO3 C-terminal stop codons, the novel truncated FMO3 Gly184Ter variant was suspected to be inactive. To easily identify the four impaired FMO3 variants (one stop codon mutation and three amino-acid substitutions) in the clinical setting, simple confirmation methods for these FMO3 variants are proposed using polymerase chain reaction/restriction fragment length polymorphism or allele-specific PCR methods. The updated whole-genome sequence data and kinetic analyses revealed that four of the seven single-nucleotide nonsense or missense FMO3 variants had moderately or severely impaired activity toward trimethylamine N-oxygenation.

Mapping interactions of calmodulin and neuronal NO synthase by crosslinking and mass spectrometry.

Neuronal nitric oxide synthase (nNOS) is a homodimeric cytochrome P450-like enzyme that catalyzes the conversion of L-arginine to nitric oxide in the presence of NADPH and molecular oxygen. The binding of calmodulin (CaM) to a linker region between the FAD/FMN-containing reductase domain, and the heme-containing oxygenase domain is needed for electron transfer reactions, reduction of the heme, and NO synthesis. Due to the dynamic nature of the reductase domain and low resolution of available full-length structures, the exact conformation of the CaM-bound active complex during heme reduction is still unresolved. Interestingly, hydrogen-deuterium exchange and mass spectrometry studies revealed interactions of the FMN domain and CaM with the oxygenase domain for iNOS, but not nNOS. This finding prompted us to utilize covalent crosslinking and mass spectrometry to clarify interactions of CaM with nNOS. Specifically, MS-cleavable bifunctional crosslinker disuccinimidyl dibutyric urea was used to identify thirteen unique crosslinks between CaM and nNOS as well as 61 crosslinks within the nNOS. The crosslinks provided evidence for CaM interaction with the oxygenase and reductase domain residues as well as interactions of the FMN domain with the oxygenase dimer. Cryo-EM studies, which gave a high-resolution model of the oxygenase domain, along with crosslink-guided docking provided a model of nNOS that brings the FMN within 15 Å of the heme in support for a more compact conformation than previously observed. These studies also point to the utility of covalent crosslinking and mass spectrometry in capturing transient dynamic conformations that may not be captured by hydrogen-deuterium exchange and mass spectrometry experiments.

Engineered Methylococcus capsulatus Bath for efficient methane conversion to isoprene.

Isoprene has numerous industrial applications, including rubber polymer and potential biofuel. Microbial methane-based isoprene production could be a cost-effective and environmentally benign process, owing to a reduced carbon footprint and economical utilization of methane. In this study, Methylococcus capsulatus Bath was engineered to produce isoprene from methane by introducing the exogenous mevalonate (MVA) pathway. Overexpression of MVA pathway enzymes and isoprene synthase from Populus trichocarpa under the control of a phenol-inducible promoter substantially improved isoprene production. M. capsulatus Bath was further engineered using a CRISPR-base editor to disrupt the expression of soluble methane monooxygenase (sMMO), which oxidizes isoprene to cause toxicity. Additionally, optimization of the metabolic flux in the MVA pathway and culture conditions increased isoprene production to 228.1 mg/L, the highest known titer for methanotroph-based isoprene production. The developed methanotroph could facilitate the efficient conversion of methane to isoprene, resulting in the sustainable production of value-added chemicals.

Genome-wide analysis of carotenoid cleavage oxygenases and identification of ripening-associated DzNCED5a in durian (Durio zibethinus) fruit.

Durian (Durio zibethinus L.), popularly known as the "King of fruits," holds significant economic importance in Southeast Asia, including Thailand. During its ripening process, the phytohormone abscisic acid (ABA) content has been reported to increase. However, a comprehensive understanding of ABA's specific role in durian fruit ripening remains elusive. Furthermore, little is known about the molecular aspects of the carotenoid cleavage pathway in this iconic fruit. Therefore, we performed genome-wide identification of the carotenoid cleavage oxygenase (CCO) family in durian. This family includes the nine-cis-epoxycarotenoid dioxygenases (NCEDs) responsible for ABA production and the carotenoid cleavage dioxygenases exhibiting diverse substrate specificities. Through phylogenetic analysis, we classified 14 CCOs in durian into 8 distinct subfamilies. Notably, each DzCCO subfamily displayed a conserved motif composition. Cis-acting element prediction showed that cis-elements related to plant hormones and environmental stress responses were distributed in the DzCCO promoter. In addition, transcriptome analysis was performed to examine the expression pattern during the fruit development and ripening stages. Interestingly, DzNCED5a, a ripening-associated gene, exhibited the highest expression level at the ripe stage, outperforming other CCOs. Its expression markedly correlated with increased ABA contents during the ripening stages of both the "Monthong" variety and other durian cultivars. Transiently expressed DzNCED5a in Nicotiana benthamiana leaves confirmed its function in ABA biosynthesis. These findings highlight the involvement of DzNCED5a in ABA production and its potential importance in durian fruit ripening. Overall, this study provides insights into the significance of CCOs in durian fruit ripening.

Expression of the two-component regulator StyS/StyR enhanced transcription of the styrene monooxygenase gene styAB and indigo biosynthesis in Escherichia coli.

Indigo, an economically important dye, could be biosynthesized from indole by catalysis of the styrene monooxygenase StyAB. To enhance indigo biosynthesis, the styAB gene and its transcription regulator gene styS/styR in styrene catabolism were cloned from Pseudomonas putida and coexpressed in Escherichia coli. The presence of the intact regulator gene styS/styR dramatically increased the transcriptional levels of styA and styB by approximately 120-fold in the recombinant strain SRAB2 with coexpression of styS/styR and styAB compared to the control strain ABST with solo expression of styAB. A yield of 67.6 mg/L indigo was detected in strain SRAB2 after 24 h of fermentation with 120 µg/mL indole, which was approximately 14-fold higher than that in the control strain ABST. The maximum yield of indigo was produced from 160 µg/mL indole in fermentation of strain SRAB2. However, the addition of styrene to the media significantly inhibited the transcription of styA and styB and consequent indigo biosynthesis in recombinant E. coli strains. Furthermore, the substitution of indole with tryptophan as the fermentation substrate remarkably boosted indigo production, and the maximal yield of 565.6 mg/L was detected in strain SRAB2 in fermentation with 1.2 mg/mL tryptophan. The results revealed that the regulation of styAB transcription by the two-component regulator StyS/StyR in styrene catabolism in P. putida was effective in E. coli, which provided a new strategy for the development of engineered E. coli strains with the capacity for highly efficient indigo production.

Rieske Oxygenase-Catalyzed Oxidative Late-Stage Functionalization during Complex Antifungal Polyketide Biosynthesis.

Rieske oxygenases (ROs) from natural product biosynthetic pathways are a poorly studied group of enzymes with significant potential as oxidative functionalization biocatalysts. A study on the ROs JerL, JerP, and AmbP from the biosynthetic pathways of jerangolid A and ambruticin VS-3 is described. Their activity was successfully reconstituted using whole-cell bioconversion systems coexpressing the ROs and their respective natural flavin-dependent reductase (FDR) partners. Feeding authentic biosynthetic intermediates and synthetic surrogates to these strains confirmed the involvement of the ROs in hydroxymethylpyrone and dihydropyran formation and revealed crucial information about the RO's substrate specificity. The pronounced dependence of JerL and JerP on the presence of a methylenolether allowed the precise temporal assignment of RO catalysis to the ultimate steps of jerangolid biosynthesis. JerP and AmbP stand out among the biosynthetic ROs studied so far for their ability to catalyze clean tetrahydropyran desaturation without further functionalizing the formed electron-rich double bonds. This work highlights the remarkable ability of ROs to highly selectively oxidize complex molecular scaffolds.

Unraveling the Valence State and Reactivity of Copper Centers in Membrane-Bound Particulate Methane Monooxygenase.

Particulate methane monooxygenase (pMMO) plays a critical role in catalyzing the conversion of methane to methanol, constituting the initial step in the C1 metabolic pathway within methanotrophic bacteria. However, the membrane-bound pMMO's structure and catalytic mechanism, notably the copper's valence state and genuine active site for methane oxidation, have remained elusive. Based on the recently characterized structure of membrane-bound pMMO, extensive computational studies were conducted to address these long-standing issues. A comprehensive analysis comparing the quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulated structures with cryo-EM data indicates that both the CuC and CuD sites tend to stay in the Cu(I) valence state within the membrane environment. Additionally, the concurrent presence of Cu(I) at both CuC and CuD sites leads to the significant reduction of the ligand-binding cavity situated between them, making it less likely to accommodate a reductant molecule such as durohydroquinone (DQH2). Subsequent QM/MM calculations reveal that the CuD(I) site is more reactive than the CuC(I) site in oxygen activation, en route to H2O2 formation and the generation of Cu(II)-O•- species. Finally, our simulations demonstrate that the natural reductant ubiquinol (CoQH2) assumes a productive binding conformation at the CuD(I) site but not at the CuC(I) site. This provides evidence that the true active site of membrane-bound pMMOs may be CuD rather than CuC. These findings clarify pMMO's catalytic mechanism and emphasize the membrane environment's pivotal role in modulating the coordination structure and the activity of copper centers within pMMO.

Characterization of a novel aromatic ring-hydroxylating oxygenase, NarA2B2, from thermophilic Hydrogenibacillus sp. strain N12.

Polycyclic aromatic hydrocarbons (PAHs) are harmful to human health due to their carcinogenic, teratogenic, and mutagenic effects. A thermophilic Hydrogenibacillus sp. strain N12 capable of degrading a variety of PAHs and derivatives was previously isolated. In this study, an aromatic ring-hydroxylating oxygenase, NarA2B2, was identified from strain N12, with substrate specificity including naphthalene, phenanthrene, dibenzothiophene, fluorene, acenaphthene, carbazole, biphenyl, and pyrene. NarA2B2 was proposed to add one or two atoms of molecular oxygen to the substrate and catalyze biphenyl at C-2, 2 or C-3, 4 positions with different characteristics than before. The key catalytic amino acids, H222, H227, and D379, were identified as playing a pivotal role in the formation of the 2-his-1-carboxylate facial triad. Furthermore, we conducted molecular docking and molecular dynamics simulations, notably, D219 enhanced the stability of the iron center by forming two stable hydrogen bonds with H222, while the mutation of F216, T223, and H302 modulated the catalytic activity by altering the pocket's size and shape. Compared to the wild-type (WT) enzyme, the degradation ratios of acenaphthene by F216A, T223A, and H302A had an improvement of 23.08%, 26.87%, and 29.52%, the degradation ratios of naphthalene by T223A and H302A had an improvement of 51.30% and 65.17%, while the degradation ratio of biphenyl by V236A had an improvement of 77.94%. The purified NarA2B2 was oxygen-sensitive when it was incubated with L-ascorbic acid in an anaerobic environment, and its catalytic activity was restored in vitro. These results contribute to a better understanding of the molecular mechanism responsible for PAHs' degradation in thermophilic microorganisms.IMPORTANCE(i) A novel aromatic ring-hydroxylating oxygenase named NarA2B2, capable of degrading multiple polycyclic aromatic hydrocarbons and derivatives, was identified from the thermophilic microorganism Hydrogenibacillus sp. N12. (ii) The degradation characteristics of NarA2B2 were characterized by adding one or two atoms of molecular oxygen to the substrate. Unlike the previous study, NarA2B2 catalyzed biphenyl at C-2, 2 or C-3, 4 positions. (iii) Catalytic sites of NarA2B2 were conserved, and key amino acids F216, D219, H222, T223, H227, V236, F243, Y300, H302, W316, F369, and D379 played pivotal roles in catalysis, as confirmed by protein structure prediction, molecular docking, molecular dynamics simulations, and point mutation.

The NADH recycling enzymes TsaC and TsaD regenerate reducing equivalents for Rieske oxygenase chemistry.

Many microorganisms use both biological and nonbiological molecules as sources of carbon and energy. This resourcefulness means that some microorganisms have mechanisms to assimilate pollutants found in the environment. One such organism is Comamonas testosteroni, which metabolizes 4-methylbenzenesulfonate and 4-methylbenzoate using the TsaMBCD pathway. TsaM is a Rieske oxygenase, which in concert with the reductase TsaB consumes a molar equivalent of NADH. Following this step, the annotated short-chain dehydrogenase/reductase and aldehyde dehydrogenase enzymes TsaC and TsaD each regenerate a molar equivalent of NADH. This co-occurrence ameliorates the need for stoichiometric addition of reducing equivalents and thus represents an attractive strategy for integration of Rieske oxygenase chemistry into biocatalytic applications. Therefore, in this work, to overcome the lack of information regarding NADH recycling enzymes that function in partnership with Rieske non-heme iron oxygenases (Rieske oxygenases), we solved the X-ray crystal structure of TsaC to a resolution of 2.18 Å. Using this structure, a series of substrate analog and protein variant combination reactions, and differential scanning fluorimetry experiments, we identified active site features involved in binding NAD+ and controlling substrate specificity. Further in vitro enzyme cascade experiments demonstrated the efficient TsaC- and TsaD-mediated regeneration of NADH to support Rieske oxygenase chemistry. Finally, through in-depth bioinformatic analyses, we illustrate the widespread co-occurrence of Rieske oxygenases with TsaC-like enzymes. This work thus demonstrates the utility of these NADH recycling enzymes and identifies a library of short-chain dehydrogenase/reductase enzyme prospects that can be used in Rieske oxygenase pathways for in situ regeneration of NADH.

Transcriptional regulation of soluble methane monooxygenase via enhancer-binding protein derived from Methylosinus sporium 5.

Methane is a major greenhouse gas, and methanotrophs regulate the methane level in the carbon cycle. Soluble methane monooxygenase (sMMO) is expressed in various methanotroph genera, including Alphaproteobacteria and Gammaproteobacteria, and catalyzes the hydroxylation of methane to methanol. It has been proposed that MmoR regulates the expression of sMMO as an enhancer-binding protein under copper-limited conditions; however, details on this transcriptional regulation remain limited. Herein, we elucidate the transcriptional pathway of sMMO depending on copper ion concentration, which affects the interaction of MmoR and sigma factor. MmoR and sigma-54 (σ54) from Methylosinus sporium 5 were successfully overexpressed in Escherichia coli and purified to investigate sMMO transcription in methanotrophs. The results indicated that σ54 binds to a promoter positioned -24 (GG) and -12 (TGC) upstream between mmoG and mmoX1. The binding affinity and selectivity are lower (Kd = 184.6 ± 6.2 nM) than those of MmoR. MmoR interacts with the upstream activator sequence (UAS) with a strong binding affinity (Kd = 12.5 ± 0.5 nM). Mutational studies demonstrated that MmoR has high selectivity to its binding partner (ACA-xx-TGT). Titration assays have demonstrated that MmoR does not coordinate with copper ions directly; however, its binding affinity to UAS decreases in a low-copper-containing medium. MmoR strongly interacts with adenosine triphosphate (Kd = 62.8 ± 0.5 nM) to generate RNA polymerase complex. This study demonstrated that the binding events of both MmoR and σ54 that regulate transcription in M. sporium 5 depend on the copper ion concentration. IMPORTANCE This study provides biochemical evidence of transcriptional regulation of soluble methane monooxygenase (sMMO) in methanotrophs that control methane levels in ecological systems. Previous studies have proposed transcriptional regulation of MMOs, including sMMO and pMMO, while we provide further evidence to elucidate its mechanism using a purified enhancer-binding protein (MmoR) and transcription factor (σ54). The characterization studies of σ54 and MmoR identified the promoter binding sites and enhancer-binding sequences essential for sMMO expression. Our findings also demonstrate that MmoR functions as a trigger for sMMO expression due to the high specificity and selectivity for enhancer-binding sequences. The UV-visible spectrum of purified MmoR suggested an iron coordination like other GAF domain, and that ATP is essential for the initiation of enhancer elements. Binding assays indicated that these interactions are blocked by the copper ion. These results provide novel insights into gene regulation of methanotrophs.

Improving the Enantioselectivity of CHMOBrevi1 for Asymmetric Synthesis of Podophyllotoxin Precursor.

(R)-ß-piperonyl-γ-butyrolactones are key building blocks for the synthesis of podophyllotoxin, which have demonstrated remarkable potential in cancer treatment. Baeyer-Villiger monooxygenases (BVMOs)-mediated asymmetric oxidation is a green approach to produce chiral lactones. While several BVMOs were able to oxidize the corresponding cyclobutanone, most BVMOs gave the (S) enantiomer while Cyclohexanone monooxygenase (CHMO) from Brevibacterium sp. HCU1 gave (R) enantiomer, but with a low enantioselectivity (75 % ee). In this study, we use a strategy called "focused rational iterative site-specific mutagenesis" (FRISM) at residues ranging from 6 Šfrom substrate. The mutations by using a restricted set of rationally chosen amino acids allow the formation of a small mutant library. By generating and screening less than 60 variants, we achieved a high ee of 96.8 %. Coupled with the cofactor regeneration system, 9.3 mM substrate was converted completely in a 100-mL scale reaction. Therefore, our work reveals a promising synthetic method for (R)-ß-piperonyl-γ-butyrolactone with the highest enantioselectivity, and provides a new opportunity for the chem-enzymatic synthesis of podophyllotoxin.

Repurposing Iron- and 2-Oxoglutarate-Dependent Oxygenases to Catalyze Olefin Hydration.

Mononuclear nonheme iron(II) and 2-oxoglutarate (Fe/2OG)-dependent oxygenases and halogenases are known to catalyze a diverse set of oxidative reactions, including hydroxylation, halogenation, epoxidation, and desaturation in primary metabolism and natural product maturation. However, their use in abiotic transformations has mainly been limited to C-H oxidation. Herein, we show that various enzymes of this family, when reconstituted with Fe(II) or Fe(III), can catalyze Mukaiyama hydration-a redox neutral transformation. Distinct from the native reactions of the Fe/2OG enzymes, wherein oxygen atom transfer (OAT) catalyzed by an iron-oxo species is involved, this nonnative transformation proceeds through a hydrogen atom transfer (HAT) pathway in a 2OG-independent manner. Additionally, in contrast to conventional inorganic catalysts, wherein a dinuclear iron species is responsible for HAT, the Fe/2OG enzymes exploit a mononuclear iron center to support this reaction. Collectively, our work demonstrates that Fe/2OG enzymes have utility in catalysis beyond the current scope of catalytic oxidation.

5-Substituted Pyridine-2,4-dicarboxylate Derivatives Have Potential for Selective Inhibition of Human Jumonji-C Domain-Containing Protein 5.

Jumonji-C domain-containing protein 5 (JMJD5) is a 2-oxoglutarate (2OG)-dependent oxygenase that plays important roles in development, circadian rhythm, and cancer through unclear mechanisms. JMJD5 has been reported to have activity as a histone protease, as an Nε-methyl lysine demethylase, and as an arginine residue hydroxylase. Small-molecule JMJD5-selective inhibitors will be useful for investigating its (patho)physiological roles. Following the observation that the broad-spectrum 2OG oxygenase inhibitor pyridine-2,4-dicarboxylic acid (2,4-PDCA) is a 2OG-competing JMJD5 inhibitor, we report that 5-aminoalkyl-substituted 2,4-PDCA derivatives are potent JMJD5 inhibitors manifesting selectivity for JMJD5 over other human 2OG oxygenases. Crystallographic analyses with five inhibitors imply induced fit binding and reveal that the 2,4-PDCA C5 substituent orients into the JMJD5 substrate-binding pocket. Cellular studies indicate that the lead compounds display similar phenotypes as reported for clinically observed JMJD5 variants, which have a reduced catalytic activity compared to wild-type JMJD5.