Functional analysis of an α-ketoglutarate-dependent non-heme iron oxygenase in fungal meroterpenoid biosynthesis.

α-Ketoglutarate-dependent non-heme iron (α-KG NHI) oxygenases compose one of the largest superfamilies of tailoring enzymes that play key roles in structural and functional diversifications. During the biosynthesis of meroterpenoids, α-KG NHI oxygenases catalyze diverse types of chemical reactions, including hydroxylation, desaturation, epoxidation, endoperoxidation, ring-cleavage, and skeletal rearrangements. Due to their catalytic versatility, keen attention has been focused on functional analyses of α-KG NHI oxygenases. This chapter provides detailed methodologies for the functional analysis of the fungal α-KG NHI oxygenase SptF, which plays an important role in the structural diversification of andiconin-derived meroterpenoids. The procedures included describe how to prepare the meroterpenoid substrate using a heterologous fungal host, measure the in vitro enzymatic activity of SptF, and how to perform structural and mutagenesis studies on SptF. These protocols are also applicable to functional analyses of other α-KG NHI oxygenases.

Photo-reduction facilitated stachydrine oxidative N-demethylation reaction: A case study of Rieske non-heme iron oxygenase Stc2 from Sinorhizobium meliloti.

Rieske-type non-heme iron oxygenases (ROs) are an important family of non-heme iron enzymes. They catalyze a diverse range of transformations in secondary metabolite biosynthesis and xenobiotic bioremediation. ROs typically shuttle electrons from NAD(P)H to the oxygenase component via reductase component(s). This chapter describes our recent biochemical characterization of stachydrine demethylase Stc2 from Sinorhizobium meliloti. In this work, the eosin Y/sodium sulfite pair serves as the photoreduction system to replace the NAD(P)H-reductase system. We describe Stc2 protein purification and quality control details as well as a flow-chemistry to separate the photo-reduction half-reaction and the oxidation half-reaction. Our study demonstrates that the eosin Y/sodium sulfite photo-reduction pair is a NAD(P)H-reductase surrogate for Stc2-catalysis in a flow-chemistry setting. Experimental protocols used in this light-driven Stc2 catalysis are likely to be applicable as a photo-reduction system for other redox enzymes.

Characterization of O2 uncoupling in biodegradation reactions of nitroaromatic contaminants catalyzed by rieske oxygenases.

Rieske oxygenases are known as catalysts that enable the cleavage of aromatic and aliphatic C-H bonds in structurally diverse biomolecules and recalcitrant organic environmental pollutants through substrate oxygenations and oxidative heteroatom dealkylations. Yet, the unproductive O2 activation, which is concomitant with the release of reactive oxygen species (ROS), is typically not taken into account when characterizing Rieske oxygenase function. Even if considered an undesired side reaction, this O2 uncoupling allows for studying active site perturbations, enzyme mechanisms, and how enzymes evolve as environmental microorganisms adapt their substrates to alternative carbon and energy sources. Here, we report on complementary methods for quantifying O2 uncoupling based on mass balance or kinetic approaches that relate successful oxygenations to total O2 activation and ROS formation. These approaches are exemplified with data for two nitroarene dioxygenases (nitrobenzene and 2-nitrotoluene dioxygenase) which have been shown to mono- and dioxygenate substituted nitroaromatic compounds to substituted nitrobenzylalcohols and catechols, respectively.

Preparation of reductases for multicomponent oxygenases.

Oxygenases catalyze crucial reactions throughout all domains of life, cleaving molecular oxygen (O2) and inserting one or two of its atoms into organic substrates. Many oxygenases, including those in the cytochrome P450 (P450) and Rieske oxygenase enzyme families, function as multicomponent systems, which require one or more redox partners to transfer electrons to the catalytic center. As the identity of the reductase can change the reactivity of the oxygenase, characterization of the latter with its cognate redox partners is critical. However, the isolation of the native redox partner or partners is often challenging. Here, we report the preparation and characterization of PbdB, the native reductase partner of PbdA, a bacterial P450 enzyme that catalyzes the O-demethylation of para-methoxylated benzoates. Through production in a rhodoccocal host, codon optimization, and anaerobic purification, this procedure overcomes conventional challenges in redox partner production and allows for robust oxygenase characterization with its native redox partner. Key lessons learned here, including the value of production in a related host and rare codon effects are applicable to a broad range of Fe-dependent oxygenases and their components.

In vitro analysis of the three-component Rieske oxygenase cumene dioxygenase from Pseudomonas fluorescens IP01.

Rieske non-heme iron-dependent oxygenases (ROs) are a versatile group of enzymes traditionally associated with the degradation of aromatic xenobiotics. In addition, ROs have been found to play key roles in natural product biosynthesis, displaying a wide catalytic diversity with typically high regio- and stereo- selectivity. However, the detailed characterization of ROs presents formidable challenges due to their complex structural and functional properties, including their multi-component composition, cofactor dependence, and susceptibility to reactive oxygen species. In addition, the substrate availability of natural product biosynthetic intermediates, the limited solubility of aromatic hydrocarbons, and the radical-mediated reaction mechanism can further complicate functional assays. Despite these challenges, ROs hold immense potential as biocatalysts for pharmaceutical applications and bioremediation. Using cumene dioxygenase (CDO) from Pseudomonas fluorescens IP01 as a model enzyme, this chapter details techniques for characterizing ROs that oxyfunctionalize aromatic hydrocarbons. Moreover, potential pitfalls, anticipated complications, and proposed solutions for the characterization of novel ROs are described, providing a framework for future RO research and strategies for studying this enzyme class. In particular, we describe the methods used to obtain CDO, from construct design to expression conditions, followed by a purification procedure, and ultimately activity determination through various activity assays.

Purification and characterization of a Rieske oxygenase and its NADH-regenerating partner proteins.

The Rieske non-heme iron oxygenases (Rieske oxygenases) comprise a class of metalloenzymes that are involved in the biosynthesis of complex natural products and the biodegradation of aromatic pollutants. Despite this desirable catalytic repertoire, industrial implementation of Rieske oxygenases has been hindered by the multicomponent nature of these enzymes and their requirement for expensive reducing equivalents in the form of a reduced nicotinamide adenine dinucleotide cosubstrate (NAD(P)H). Fortunately, however, some Rieske oxygenases co-occur with accessory proteins, that through a downstream reaction, recycle the needed NAD(P)H for catalysis. As these pathways and accessory proteins are attractive for bioremediation applications and enzyme engineering campaigns, herein, we describe methods for assembling Rieske oxygenase pathways in vitro. Further, using the TsaMBCD pathway as a model system, in this chapter, we provide enzymatic, spectroscopic, and crystallographic methods that can be adapted to explore both Rieske oxygenases and their co-occurring accessory proteins.

Expression, purification and characterization of non-heme iron-dependent mono-oxygenase OzmD in oxazinomycin biosynthesis.

Oxazinomycin is a C-nucleoside natural product characterized by a 1,3-oxazine ring linked to ribose via a C-C glycosidic bond. Construction of the 1,3-oxazine ring depends on the activity of OzmD, which is a mononuclear non-heme iron-dependent enzyme from a family of enzymes that contain a domain of unknown function (DUF) 4243. OzmD catalyzes an unusual oxidative ring rearrangement of a pyridine derivative that releases cyanide as a by-product in the final stage of oxazinomycin biosynthesis. The intrinsic sensitivity of the OzmD substrate to oxygen along with the oxygen dependency of catalysis presents significant challenges in conducting in vitro enzymatic assays. This chapter describes the detailed procedures that have been used to characterize OzmD, including protein preparation, activity assays, and reaction by-product identification.

Observing extradiol dioxygenases in action through a crystalline lens.

Extradiol dioxygenases are a class of non-heme iron-dependent enzymes found in eukaryotes and prokaryotes that play a vital role in the aerobic catabolism of aromatic compounds. They are generally divided into three evolutionarily independent superfamilies with different protein folds. Our recent studies have shed light on the catalytic mechanisms and structure-function relationships of two specific extradiol dioxygenases: 3-hydroxyanthranilate-3,4-dioxygenase, a Type III enzyme essential in mammals for producing a precursor for nicotinamide adenine dinucleotide, and L-3,4-dihydroxyphenylalanine dioxygenase, an uncommon form of Type I enzymes involved in natural product biosynthesis. This work details the expression and isolation methods for these extradiol dioxygenases and introduces approaches to achieve homogeneity and high occupancy of the enzyme metal centers. Techniques such as ultraviolet-visible and electron paramagnetic resonance spectroscopies, as well as oxygen electrode measurements, are discussed for probing the interaction of the non-heme iron center with ligands and characterizing enzymatic activities. Moreover, protein crystallization has been demonstrated as a powerful tool to study these enzymes. We highlight in crystallo reactions and single-crystal spectroscopic methods to further elucidate enzymatic functions and protein dynamics.

Methods used to determine the structure of the oxygenase component of naphthalene 1,2 dioxygenase.

Rieske non-heme iron oxygenases are ubiquitously expressed in prokaryotes. These enzymes catalyze a wide variety of reactions, including cis-dihydroxylation, mono-hydroxylation, sulfoxidation, and demethylation. They contain a Rieske-type [2Fe-2S] cluster and an active site with a mono-nuclear iron bound to a 2-His carboxylate triad. Naphthalene 1,2 dioxygenase, a representative of this family, catalyzes the conversion of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. This transformation requires naphthalene, two electrons, and an oxygen molecule. The first structure of the terminal oxygenase component of a Rieske non-heme iron oxygenase to be determined was naphthalene 1,2 dioxygenase (NDO-O). In this article, we describe in detail the methods used to recombinantly express and purify NDO-O in rich and minimal salts media, the crystallization of NDO-O for structure determination by X-ray crystallography, the challenges faced, and the methods used for the preparation of enzyme ligand complexes. The methods used here resulted in the determination of several NDO-O complexes with aromatic substrates, nitric oxide, oxygen molecule, and products, leading to an initial understanding of the mechanism of enzyme catalysis and the molecular determinants of the regio- and stereo-specificity of this class of enzymes.

Computational Insights into the Non-Heme Diiron Alkane Monooxygenase Enzyme AlkB: Electronic Structures, Dioxygen Activation, and Hydroxylation Mechanism of Liquid Alkanes.

Alkane monooxygenase (AlkB) is a membrane-spanning metalloenzyme that catalyzes the terminal hydroxylation of straight-chain alkanes involved in the microbially mediated degradation of liquid alkanes. According to the cryoEM structures, AlkB features a unique multihistidine ligand coordination environment with a long Fe-Fe distance in its active center. Up to now, how AlkB employs the diiron center to activate dioxygen and which species is responsible for triggering the hydroxylation are still elusive. In this work, we constructed computational models and performed quantum mechanics/molecular mechanics (QM/MM) calculations to illuminate the electronic characteristics of the diiron active center and how AlkB carries out the terminal hydroxylation. Our calculations revealed that the spin-spin interaction between two irons is rather weak. The dioxygen may ligate to either the Fe1 or Fe2 atom and prefers to act as a linker to increase the spin-spin interaction of two irons, facilitating the dioxygen cleavage to generate the highly reactive Fe(IV)═O. Thus, AlkB employs Fe(IV)═O to trigger the hydrogen abstraction. In addition, the previously suggested mechanism that AlkB uses both the dioxygen and Fe-coordinated water to perform hydroxylation was calculated to be unlikely. Besides, our results indicate that AlkB cannot use the Fe-coordinated dioxygen to directly trigger hydrogen abstraction.

Structural and Functional Characterization of a Novel Class A Flavin Monooxygenase from Bacillus niacini.

A gene cluster responsible for the degradation of nicotinic acid (NA) in Bacillus niacini has recently been identified, and the structures and functions of the resulting enzymes are currently being evaluated to establish pathway intermediates. One of the genes within this cluster encodes a flavin monooxygenase (BnFMO) that is hypothesized to catalyze a hydroxylation reaction. Kinetic analyses of the recombinantly purified BnFMO suggest that this enzyme catalyzes the hydroxylation of 2,6-dihydroxynicotinic acid (2,6-DHNA) or 2,6-dihydroxypyridine (2,6-DHP), which is formed spontaneously by the decarboxylation of 2,6-DHNA. To understand the details of this hydroxylation reaction, we determined the structure of BnFMO using a multimodel approach combining protein X-ray crystallography and cryo-electron microscopy (cryo-EM). A liganded BnFMO cryo-EM structure was obtained in the presence of 2,6-DHP, allowing us to make predictions about potential catalytic residues. The structural data demonstrate that BnFMO is trimeric, which is unusual for Class A flavin monooxygenases. In both the electron density and coulomb potential maps, a region at the trimeric interface was observed that was consistent with and modeled as lipid molecules. High-resolution mass spectral analysis suggests that there is a mixture of phosphatidylethanolamine and phosphatidylglycerol lipids present. Together, these data provide insights into the molecular details of the central hydroxylation reaction unique to the aerobic degradation of NA in Bacillus niacini.

Functional and spectroscopic approaches to determining thermal limitations of Rieske oxygenases.

The biotechnological potential of Rieske Oxygenases (ROs) and their cognate reductases remains unmet, in part because these systems can be functionally short-lived. Here, we describe a set of experiments aimed at identifying both the functional and structural stability limitations of ROs, using terephthalate (TPA) dioxygenase (from Comamonas strain E6) as a model system. Successful expression and purification of a cofactor-complete, histidine-tagged TPA dioxygenase and reductase protein system requires induction with the Escherichia coli host at stationary phase as well as a chaperone inducing cold-shock and supplementation with additional iron, sulfur, and flavin. The relative stability of the Rieske cluster and mononuclear iron center can then be assessed using spectroscopic and functional measurements following dialysis in an iron chelating buffer. These experiments involve measurements of the overall lifetime of the system via total turnover number using both UV-Visible absorbance and HPLC analyses, as well specific activity as a function of temperature. Important methods for assessing the stability of these multi-cofactor, multi-protein dependent systems at multiple levels of structure (secondary to quaternary) include differential scanning calorimetry, circular dichroism, and metallospectroscopy. Results can be rationalized in terms of three-dimensional structures and bioinformatics. The experiments described here provide a roadmap to a detailed characterization of the limitations of ROs. With a few notable exceptions, these issues are not widely addressed in current literature.

Semi-rational design based on the interaction between SmFMO and FAD isoalloxazine ring to enhance the enzyme activity.

Flavin monooxygenases (FMOs) have been widely used in the biosynthesis of natural compounds due to their excellent stereoselectivity, regioselectivity and chemoselectivity. Stenotrophomonas maltophilia flavin monooxygenase (SmFMO) has been reported to catalyze the oxidation of various thiols to corresponding sulfoxides, but its activity is relatively low. Herein, we obtained a mutant SmFMOF52G which showed 4.35-fold increase in kcat/Km (4.96 mM-1s-1) and 6.84-fold increase in enzyme activity (81.76 U/g) compared to the SmFMOWT (1.14 mM-1s-1 and 11.95 U/g) through semi-rational design guided by structural analysis and catalytic mechanism combined with high-throughput screening. By forming hydrogen bond with O4 atom of FAD isoalloxazine ring and reducing steric hindrance, the conformation of FAD isoalloxazine ring in SmFMOF52G is more stable, and NADPH and substrate are closer to FAD isoalloxazine ring, shortening the distances of hydrogen transfer and substrate oxygenation, thereby increasing the rate of reduction and oxidation reactions and enhancing enzyme activity. Additionally, the overall structural stability and substrate binding capacity of the SmFMOF52G have significant improved than that of SmFMOWT. The strategy used in this study to improve the enzyme activity of FMOs may have generality, providing important references for the rational and semi-rational engineering of FMOs.

Asymmetric Sulfoxidations Catalyzed by Bacterial Flavin-Containing Monooxygenases.

Flavin-containing monooxygenase from Methylophaga sp. (mFMO) was previously discovered to be a valuable biocatalyst used to convert small amines, such as trimethylamine, and various indoles. As FMOs are also known to act on sulfides, we explored mFMO and some mutants thereof for their ability to convert prochiral aromatic sulfides. We included a newly identified thermostable FMO obtained from the bacterium Nitrincola lacisaponensis (NiFMO). The FMOs were found to be active with most tested sulfides, forming chiral sulfoxides with moderate-to-high enantioselectivity. Each enzyme variant exhibited a different enantioselective behavior. This shows that small changes in the substrate binding pocket of mFMO influence selectivity, representing a tunable biocatalyst for enantioselective sulfoxidations.

Engineering of Rieske dioxygenase variants with improved cis-dihydroxylation activity for benzoates.

Rieske dioxygenases have a long history of being utilized as green chemical tools in the organic synthesis of high-value compounds, due to their capacity to perform the cis-dihydroxylation of a wide variety of aromatic substrates. The practical utility of these enzymes has been hampered however by steric and electronic constraints on their substrate scopes, resulting in limited reactivity with certain substrate classes. Herein, we report the engineering of a widely used member of the Rieske dioxygenase class of enzymes, toluene dioxygenase (TDO), to produce improved variants with greatly increased activity for the cis-dihydroxylation of benzoates. Through rational mutagenesis and screening, TDO variants with substantially improved activity over the wild-type enzyme were identified. Homology modeling, docking studies, molecular dynamics simulations, and substrate tunnel analysis were applied in an effort to elucidate how the identified mutations resulted in improved activity for this polar substrate class. These analyses revealed modification of the substrate tunnel as the likely cause of the improved activity observed with the best-performing enzyme variants.

Discovery of a new class of bacterial heme-containing CC cleaving oxygenases.

Previously, some bacteria were shown to harbour enzymes capable of catalysing the oxidative cleavage of the double bond of t-anethole and related compounds. The cofactor dependence of these enzymes remained enigmatic due to a lack of biochemical information. We report on catalytic and structural details of a representative of this group of oxidative enzymes: t-anethole oxygenase from Stenotrophomonas maltophilia (TAOSm). The bacterial enzyme could be recombinantly expressed and purified, enabling a detailed biochemical study that has settled the dispute on its cofactor dependence. We have established that TAOSm contains a tightly bound b-type heme and merely depends on dioxygen for catalysis. It was found to accept t-anethole, isoeugenol and O-methyl isoeugenol as substrates, all being converted into the corresponding aromatic aldehydes without the need of any cofactor regeneration. The elucidated crystal structure of TAOSm has revealed that it contains a unique active site architecture that is conserved for this distinct class of heme-containing bacterial oxygenases. Similar to other hemoproteins, TAOSm has a histidine (His121) as proximal ligand. Yet, unique for TAOs, an arginine (Arg89) is located at the distal axial position. Site directed mutagenesis confirmed crucial roles for these heme-liganding residues and other residues that form the substrate binding pocket. In conclusion, the results reported here reveal a new class of bacterial heme-containing oxygenases that can be used for the cleavage of alkene double bonds, analogous to ozonolysis in organic chemistry.

Mechanisms for Methane and Ammonia Oxidation by Particulate Methane Monooxygenase.

Particulate MMO (pMMO) catalyzes the oxidation of methane to methanol and also ammonia to hydroxylamine. Experimental characterization of the active site has been very difficult partly because the enzyme is membrane-bound. However, recently, there has been major progress mainly through the use of cryogenic electron microscopy (cryoEM). Electron paramagnetic resonance (EPR) and X-ray spectroscopy have also been employed. Surprisingly, the active site has only one copper. There are two histidine ligands and one asparagine ligand, and the active site is surrounded by phenyl alanines but no charged amino acids in the close surrounding. The present study is the first quantum chemical study using a model of that active site (CuD). Low barrier mechanisms have been found, where an important part is that there are two initial proton-coupled electron transfer steps to a bound O2 ligand before the substrate enters. Surprisingly, this leads to large radical character for the oxygens even though they are protonated. That result is very important for the ability to accept a proton from the substrates. Methods have been used which have been thoroughly tested for redox enzyme mechanisms.

An Unusual Ferryl Intermediate and Its Implications for the Mechanism of Oxacyclization by the Loline-Producing Iron(II)- and 2-Oxoglutarate-Dependent Oxygenase, LolO.

N-Acetylnorloline synthase (LolO) is one of several iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases that catalyze sequential reactions of different types in the biosynthesis of valuable natural products. LolO hydroxylates C2 of 1-exo-acetamidopyrrolizidine before coupling the C2-bonded oxygen to C7 to form the tricyclic loline core. Each reaction requires cleavage of a C-H bond by an oxoiron(IV) (ferryl) intermediate; however, different carbons are targeted, and the carbon radicals have different fates. Prior studies indicated that the substrate-cofactor disposition (SCD) controls the site of H· abstraction and can affect the reaction outcome. These indications led us to determine whether a change in SCD from the first to the second LolO reaction might contribute to the observed reactivity switch. Whereas the single ferryl complex in the C2 hydroxylation reaction was previously shown to have typical Mössbauer parameters, one of two ferryl complexes to accumulate during the oxacyclization reaction has the highest isomer shift seen to date for such a complex and abstracts H· from C7 ∼ 20 times faster than does the first ferryl complex in its previously reported off-pathway hydroxylation of C7. The detectable hydroxylation of C7 in competition with cyclization by the second ferryl complex is not enhanced in 2H2O solvent, suggesting that the C2 hydroxyl is deprotonated prior to C7-H cleavage. These observations are consistent with the coordination of the C2 oxygen to the ferryl complex, which may reorient its oxo ligand, the substrate, or both to positions more favorable for C7-H cleavage and oxacyclization.

Fine-tuning an aromatic ring-hydroxylating oxygenase to degrade high molecular weight polycyclic aromatic hydrocarbon.

Rieske nonheme iron aromatic ring-hydroxylating oxygenases (RHOs) play pivotal roles in determining the substrate preferences of polycyclic aromatic hydrocarbon (PAH) degraders. However, their potential to degrade high molecular weight PAHs (HMW-PAHs) has been relatively unexplored. NarA2B2 is an RHO derived from a thermophilic Hydrogenibacillus sp. strain N12. In this study, we have identified four "hotspot" residues (V236, Y300, W316, and L375) that may hinder the catalytic capacity of NarA2B2 when it comes to HMW-PAHs. By employing structure-guided rational enzyme engineering, we successfully modified NarA2B2, resulting in NarA2B2 variants capable of catalyzing the degradation of six different types of HMW-PAHs, including pyrene, fluoranthene, chrysene, benzo[a]anthracene, benzo[b]fluoranthene, and benzo[a]pyrene. Three representative variants, NarA2B2W316I, NarA2B2Y300F-W316I, and NarA2B2V236A-W316I-L375F, not only maintain their abilities to degrade low-molecular-weight PAHs (LMW-PAHs) but also exhibited 2 to 4 times higher degradation efficiency for HMW-PAHs in comparison to another isozyme, NarAaAb. Computational analysis of the NarA2B2 variants predicts that these modifications alter the size and hydrophobicity of the active site pocket making it more suitable for HMW-PAHs. These findings provide a comprehensive understanding of the relationship between three-dimensional structure and functionality, thereby opening up possibilities for designing improved RHOs that can be more effectively used in the bioremediation of PAHs.

Engineering a Carotenoid Cleavage Oxygenase for Coenzyme-Free Synthesis of Vanillin from Ferulic Acid.

One-pot biosynthesis of vanillin from ferulic acid without providing energy and cofactors adds significant value to lignin waste streams. However, naturally evolved carotenoid cleavage oxygenase (CCO) with extreme catalytic conditions greatly limited the above pathway for vanillin bioproduction. Herein, CCO from Thermothelomyces thermophilus (TtCCO) was rationally engineered for achieving high catalytic activity under neutral pH conditions and was further utilized for constructing a one-pot synthesis system of vanillin with Bacillus pumilus ferulic acid decarboxylase. TtCCO with the K192N-V310G-A311T-R404N-D407F-N556A mutation (TtCCOM3) was gradually obtained using substrate access channel engineering, catalytic pocket engineering, and pocket charge engineering. Molecular dynamics simulations revealed that reducing the site-blocking effect in the substrate access channel, enhancing affinity for substrates in the catalytic pocket, and eliminating the pocket's alkaline charge contributed to the high catalytic activity of TtCCOM3 under neutral pH conditions. Finally, the one-pot synthesis of vanillin in our study could achieve a maximum rate of up to 6.89 ± 0.3 mM h-1. Therefore, our study paves the way for a one-pot biosynthetic process of transforming renewable lignin-related aromatics into valuable chemicals.