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Fungal polysaccharide monooxygenases (PMOs) oxidatively degrade cellulose and other carbohydrate polymers via a mononuclear copper active site using either O 2 or H 2 O 2 as a cosubstrate. Cellulose-active fungal PMOs in the auxiliary activity 9 (AA9) family have a conserved second-sphere hydrogen-bonding network consisting of histidine, glutamine, and tyrosine residues. The second-sphere histidine has been hypothesized to play a role in proton transfer in the O 2 -dependent PMO reaction. Here the role of the second-sphere histidine (H157) in an AA9 PMO, Mt PMO9E, was investigated. This PMO is active on soluble cello-oligosaccharides such as cellohexaose (Glc6), thus enabling kinetic analysis with the point variants H157A and H157Q. The variants appeared to fold similarly to the wild-type (WT) enzyme and yet exhibited weaker affinity toward Glc6 than WT (WT K D = 20 ± 3 μM). The variants had comparable oxidase (O 2 reduction to H 2 O 2 ) activity to WT at all pH values tested. Using O 2 as a cosubstrate, the variants were less active for Glc6 hydroxylation than WT, with H157A being the least active. Similarly, H157Q was competent for Glc6 hydroxylation with H 2 O 2 , but H157A was less active. Comparison of the crystal structures of H157Q and WT Mt PMO9E reveals that a terminal heteroatom of Q157 overlays with N ε of H157. Altogether, the data suggest that H157 is not important for proton transfer, but support a role for H157 as a hydrogen-bond donor to diatomic-oxygen intermediates, thus facilitating catalysis with either O 2 or H 2 O 2 .

The use of light as abundant, renewable, and clean energy source to boost lytic polysaccharide monooxygenase (LPMO) reactions represents an exciting and yet under-explored opportunity. Herein we demonstrated that photosensitizers, commonly used in photodynamic therapy, which act through the photocatalytic Type I mechanism can drive the oxidation of PASC by LPMOs, whereas Type II photosensitizers are not capable of promoting the LPMO activity. We analyzed Type I and Type II photosensitizers (methylene blue and tetraiodide salt of meso-tetrakis-(4-N-methylpyridyl) porphyrin, respectively) and demonstrated that, even without an addition of external reductant, Type I was capable of boosting Thermothelomyces thermophila MtLPMO9A activity in the presence of light. We also evaluated the photobiosystem in the presence and/or absence of molecular oxygen (O 2 ) and hydrogen peroxide (H 2 O 2 ), and investigated the role of superoxide radical in the methylene blue fueled reactions. Furthermore, we demonstrated that sodium bisulfite (NaHSO 3 ), a chemical scavenger of H 2 O 2 , acts by safeguarding the enzyme from oxidative damage caused by accumulation of H 2 O 2 early in photosensitizer-driven LPMO reactions. Finally, the results of the present work demonstrated that light-driven LPMO reactions mediated photodynamic therapy (PDT) Type I photosensitizers, which also includes molecules such as curcumin and riboflavin, is a general phenomenon., (© 2024 Wiley-VCH GmbH.)

Brassica napus is a well-known allopolyploid oil crop with high commercial potential. Gibberellin oxidase (GAox) is an essential enzyme that activates gibberellins, which regulate plant growth, and development, and have a significant impact on plant responses to abiotic stress. However, the comprehensive understanding of GAox genes and their evolution in Brassica plants remains elusive. Using advanced bioinformatics tools, this study identified 125 candidate GAox genes from the whole genomes of three key Brassica species. This study also investigated sequence characteristics, conserved motifs, exon/intron structures, cis-acting elements, syntenic analysis, duplication events and expression patterns. Subcellular localization analysis showed that the BnGA2ox14 and BnGA2ox15 proteins are located in the nucleus, whereas BnGA2ox26 is specifically localized to the chloroplast. Yeast one-hybrid and dual-luciferase assays demonstrated that MYELOCYTOMATOSIS 4 (BnMYC4) and ABA-INDUCIBLE BHLH-TYPE TRANSCRIPTION FACTOR (BnAIB) bind to the BnGA2ox15 promoter and activate its transcription. Molecular docking analysis further elucidated their interaction structures and identified potential binding sites. Roots transformations show that overexpression of BnGA2ox15 increased sensitivity to PEG-6000 treatment in rapeseed. In brief, this study reveals that BnGA2ox15 is a downstream target in JA and ABA signaling pathways, functioning as a negative regulator in response to drought stress., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lytic polysaccharide monooxygenases (LPMOs) are one of the emerging classes of copper metalloenzymes that have received considerable attention due to their ability to boost the enzymatic conversion of intractable polysaccharides such as plant cell walls and chitin polymers. LPMOs catalyze the oxidative cleavage of β-1,4-glycosidic bonds using molecular O 2 or H 2 O 2 in the presence of an external electron donor. LPMOs have been classified as an auxiliary active (AA) class of enzymes and, further based on substrate specificity, divided into eight families. Until now, multiple LPMOs from AA9 and AA10 families, mostly from microbial sources, have been investigated; the exact mechanism and structure-function are elusive to date, and recently discovered AA families of LPMOs are just scratched. This review highlights the origin and discovery of the enzyme, nomenclature, three-dimensional protein structure, substrate specificity, copper-dependent reaction mechanism, and different techniques used to determine the product formation through analytical and biochemical methods. Moreover, the diverse functions of proteins in various biological activities such as plant-pathogen/pest interactions, cell wall remodeling, antibiotic sensitivity of biofilms, and production of nanocellulose along with certain obstacles in deconstructing the complex polysaccharides have also been summarized, while highlighting the innovative and creative ways to overcome the limitations of LPMOs in hydrolyzing the biomass., (© 2024 Federation of European Biochemical Societies.)

Alpha-ketoglutarate-dependent dioxygenases (αKGDs) have recently emerged as useful biocatalysts for C-H oxidation and functionalization. In this work, we characterized a new αKGD from aculene biosynthesis, AneA, which displays broad promiscuity toward a number of substrates with different ring systems. Unexpectedly, AneA was found to be capable of both desaturation and hydroxylation and require an amino ester motif on its substrate for productive catalysis. Insights gathered from the functional characterization and substrate-activity profiling of AneA enabled the development of a chemoenzymatic strategy toward several complex sesquiterpenoids., (© 2024 Wiley-VCH GmbH.)

Sulfur is an essential element for life. Bacteria can obtain sulfur from inorganic sulfate; but in the sulfur starvation-induced response, Pseudomonads employ two-component flavin-dependent monooxygenases (TC-FMOs) from the msu and sfn operons to assimilate sulfur from environmental compounds including alkanesulfonates and dialkylsulfones. Here, we report binding studies of oxidized FMN to enzymes involved within the P. fluorescens enzymatic pathway responsible for converting dimethylsulfone (DMSO 2 ) to sulfite. In this catabolic pathway, SfnG serves as the initial TC-FMO for sulfur assimilation, which is investigated in detail by solving the 2.6-Å resolution crystal structure of unliganded SfnG and the 1.75-Å resolution crystal structure of the SfnG ternary complex containing FMN and DMSO 2 . We find that SfnG adopts a (β/α) 8 barrel fold with a distinct quaternary configuration from other tetrameric class C TC-FMOs. To probe the unexpected tetramer arrangement, structural heterogeneity is assessed by chromatography and light scattering to confirm ligand binding correlates with a tetramer. Binding of FMN and DMSO 2 accompanies ordering of the active site, with DMSO 2 bound on the si -face of the flavin. A previously unobserved protein backbone conformation is found within the oxygen-binding site on the re -face of the flavin. Functional assays and the positioning of ligands with respect to the oxygen-binding site are consistent with use of an N5-(hydro)peroxyflavin pathway. Biochemical endpoint assays and docking studies reveal SfnG breaks the C-S bond of a range of dialkylsulfones., Competing Interests: Competing interests statement:The authors declare no competing interest.

Lytic polysaccharide monooxygenases (LPMOs) form a copper-dependent family of enzymes classified under the auxiliary activity (AA) superfamily. The LPMOs are known for their boosting of polysaccharide degradation through oxidation of the glycosidic bonds that link the monosaccharide subunits. This oxidation has been proposed to be dependent on either O 2 or H 2 O 2 as cosubstrate. Theoretical investigations have previously supported both mechanisms, although this contrasts with recent experiments. A possible explanation is that the theoretical results critically depend on how the Cu active site is modeled. This has also led to different results even when employing only H 2 O 2 as cosubstrate. In this paper, we investigate both the O 2 - and H 2 O 2 -driven pathways, employing Ls AA9 as the underlying LPMO and a theoretical model based on a quantum mechanics/molecular mechanics (QM/MM) framework. We ensure to consistently include all residues known to be important by using extensive QM regions of up to over 900 atoms. We also investigate several conformers that can partly explain the differences seen in previous studies. We find that the O 2 -driven reaction is unfeasible, in contrast with our previous QM/MM calculations with smaller QM regions. Meanwhile, the H 2 O 2 -driven pathway is feasible, showing that for Ls AA9, only H 2 O 2 is a viable cosubstrate as proposed experimentally.

Biocatalysis can be powerful in organic synthesis but is often limited by enzymes' substrate scope and selectivity. Developing a biocatalytic step involves identifying an initial enzyme for the target reaction followed by optimization through rational design, directed evolution, or both. These steps are time consuming, resource-intensive, and require expertise beyond typical organic chemistry. Thus, an effective strategy for streamlining the process from enzyme identification to implementation is essential to expanding biocatalysis. Here, we present a strategy combining bioinformatics-guided enzyme mining and ancestral sequence reconstruction (ASR) to resurrect enzymes for biocatalytic synthesis. Specifically, we achieve an enantioselective synthesis of azaphilone natural products using two ancestral enzymes: a flavin-dependent monooxygenase (FDMO) for stereodivergent oxidative dearomatization and a substrate-selective acyltransferase (AT) for the acylation of the enzymatically installed hydroxyl group. This cascade, stereocomplementary to established chemoenzymatic routes, expands access to enantiomeric linear tricyclic azaphilones. By leveraging the co-occurrence and coevolution of FDMO and AT in azaphilone biosynthetic pathways, we identified an AT candidate, CazE, and addressed its low solubility and stability through ASR, obtaining a more soluble, stable, promiscuous, and reactive ancestral AT (AncAT). Sequence analysis revealed AncAT as a chimeric composition of its descendants with enhanced reactivity likely due to ancestral promiscuity. Flexible receptor docking and molecular dynamics simulations showed that the most reactive AncAT promotes a reactive geometry between substrates. We anticipate that our bioinformatics-guided, ASR-based approach can be broadly applied in target-oriented synthesis, reducing the time required to develop biocatalytic steps and efficiently access superior biocatalysts.

In this study, we developed a wearable nanozyme-enzyme electrochemical biosensor that enablies sweat lactate monitoring. The biosensor comprises a flexible electrode system prepared on a polyimide (PI) film and the Janus textile for unidirectional sweat transport. We obtained favorable electrochemical activities for hydrogen peroxide reduction by modifying the laser-scribed graphene (LSG) electrode with cerium dioxide (CeO 2 )-molybdenum disulphide (MoS 2 ) nanozyme and gold nanoparticles (AuNPs). By further immobilisation of lactate oxidase (LOx), the proposed biosensor achieves chronoamperometric lactate detection in artificial sweat within a range of 0.1-50.0 mM, a high sensitivity of 25.58 μA mM -1 cm -2 and a limit of detection (LoD) down to 0.135 mM, which fully meets the requirements of clinical diagnostics. We demonstrated accurate lactate measurements in spiked artificial sweat, which is consistent with standard ELISA results. To monitor the sweat produced by volunteers while exercising, we conducted on-body tests, showcasing the wearable biosensor's ability to provide clinical sweat lactate diagnosis for medical treatment and sports management., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidatively cleave recalcitrant polysaccharides such as cellulose. Several studies have reported LPMO action in synergy with other carbohydrate-active enzymes (CAZymes) for the degradation of lignocellulosic biomass but direct LPMO action at the plant tissue level remains challenging to investigate. Here, we have developed a MALDI-MS imaging workflow to detect oxidised oligosaccharides released by a cellulose-active LPMO at cellular level on maize tissues. Using this workflow, we imaged LPMO action and gained insight into the spatial variation and relative abundance of oxidised and non-oxidised oligosaccharides. We reveal a targeted action of the LPMO related to the composition and organisation of plant cell walls., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Exploring the structural basis of membrane proteins is significant for a deeper understanding of protein functions. In situ analysis of membrane proteins and their dynamics, however, still challenges conventional techniques. Here we report the first attempt to immobilize membrane protein complexes on surface-enhanced Raman scattering (SERS)-active supports, titanium dioxide-coated silver (Ag@TiO 2 ) nanoparticles. Biocompatible immobilization of microsomal monooxygenase complexes is achieved through lipid fission and fusion. SERS activity of the Ag@TiO 2 nanoparticles enables in situ monitoring of protein-protein electron transfer and enzyme catalysis in real time. Through SERS fingerprints of the monooxygenase redox centers, the correlations between these protein-ligand interactions and reactive oxygen species generation are revealed, providing novel insights into the molecular mechanisms underlying monooxygenase-mediated apoptotic regulation. This study offers a novel strategy to explore structure-function relationships of membrane protein complexes and has the potential to advance the development of novel reactive oxygen species-inducing drugs for cancer therapy.

Fungal unspecific peroxygenases (UPOs) are gaining momentum in synthetic chemistry. Of special interest is the UPO from Marasmius rotula ( Mro UPO), which shows an exclusive repertoire of oxyfunctionalizations, including the terminal hydroxylation of alkanes, the α-oxidation of fatty acids and the C-C cleavage of corticosteroids. However, the lack of heterologous expression systems to perform directed evolution has impeded its engineering for practical applications. Here, we introduce a close ortholog of Mro UPO, a UPO gene from Marasmius wettsteinii ( Mwe UPO-1), that has a similar reaction profile to Mro UPO and for which we have set up a directed evolution platform based on tandem-yeast expression. Recombinant Mwe UPO-1 was produced at high titers in the bioreactor (0.7 g/L) and characterized at the biochemical and atomic levels. The conjunction of soaking crystallographic experiments at a resolution up to 1.6 Å together with the analysis of reaction patterns sheds light on the substrate preferences of this promiscuous biocatalyst.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes which are categorized in the CAZy database under auxiliary activities families AA9-11, 13, 14-17. Secreted by various microorganisms, they play a crucial role in carbon recycling, particularly in fungal saprotrophs. LPMOs oxidize polysaccharides through monooxygenase/peroxygenase activities and exhibit peroxidase and oxidase activities, with variations among different families. AA16, a newly identified LPMO family, is noteworthy due to limited studies on its members, thus rendering the characterization of AA16 enzymes vital for addressing controversies around their functions. This study focused on heterologous expression and biochemical study of an AA16 LPMO from Thermothelomyces thermophilus (formerly known as Myceliophthora thermophila), namely MtLPMO16A. Substrate specificity evaluation of MtLPMO16A showed oxidative cleavage of hemicellulosic substrates and no activity on cellulose, accompanied by a strong oxidase activity. A comparative analysis with an LPMO from AA9 family explored correlations between these families, while MtLPMO16A was shown to boost the activity of some AA9 family LPMOs. The results offer new insights into the AA16 family's action mode and microbial hemicellulose decomposition mechanisms in nature., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Antonis Karantonis reports financial support was provided by Hellenic Foundation for Research and Innovation. Evangelos Topakas reports financial support was provided by European Regional Development Fund of the European Union and Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Bioactive dimeric (pre-)anthraquinones are ubiquitous in nature and are found in bacteria, fungi, insects, and plants. Their biosynthesis via oxidative phenol coupling (OPC) is catalyzed by cytochrome P450 enzymes, peroxidases, or laccases. While the biocatalysis of OPC in molds (Ascomycota) is well-known, the respective enzymes in mushroom-forming fungi (Basidiomycota) are unknown. Here, we report on the biosynthesis of the atropisomers phlegmacin A 1 and B 1 of the mushroom Cortinarius odorifer. The biosynthesis of these unsymmetrically 7,10'-homo-coupled dihydroanthracenones was heterologously reconstituted in the mold Aspergillus niger. Methylation of the parental monomer atrochrysone to its 6-O-methyl ether torosachrysone by the O-methyltransferase CoOMT1 precedes the regioselective homocoupling to phlegmacin, catalyzed by the enzyme CoUPO1 annotated as an "unspecific peroxygenase" (UPO). Our results reveal an unprecedented UPO reaction, thereby expanding the biocatalytic portfolio of oxidative phenol coupling beyond the commonly reported enzymes. The results show that Basidiomycota use peroxygenases to selectively couple aryls independently of and convergently to any other group of organisms, emphasizing the central role of OPC in natural processes., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Unspecific peroxygenase from Agrocybe aegerite (AaeUPO) is a remarkable catalyst for the oxyfunctionalization of non-activated C-H bonds under mild conditions. It exhibits comparable activity to P450 monooxygenase but offers the advantage of using H 2 O 2 instead of a complex electron transport chain to reductively activate O 2 . Here, we demonstrate the successful oxidation of cyclohexane to cyclohexanol/cyclohexanone (KA-oil) using sol-gel encapsulated AaeUPO. Remarkably, cyclohexane serves both as a solvent and a substrate in this system, which simplifies product isolation. The ratio of cyclohexanone to cyclohexanol using this approach is remarkably higher compared to the oxidation using free AaeUPO in aqueous media using acetonitrile as a cosolvent. The utilization of sol-gel encapsulated AaeUPO offers a promising approach for oxyfunctionalization reactions and improves the chances for this enzyme to be incorporated in the same pot with other chemical transformations., (© 2024 The Authors. ChemistryOpen published by Wiley-VCH GmbH.)

S-omeprazole and R-rabeprazole are important proton pump inhibitors (PPIs) used for treating peptic disorders. They can be biosynthesized from the corresponding sulfide catalyzed by Baeyer-Villiger monooxygenases (BVMOs). During the development of BVMOs for target sulfoxide preparation, stereoselectivity and overoxidation degree are important factors considered most. In the present study, LnPAMO-Mu15 designed previously and TtPAMO from Thermothelomyces thermophilus showed high (S)- and (R)-configuration stereoselectivity respectively towards thioethers. TtPAMO was found to be capable of oxidating omeprazole sulfide (OPS) and rabeprazole sulfide (RPS) into R-omeprazole and R-rabeprazole respectively. However, the overoxidation issue existed and limited the application of TtPAMO in the biosynthesis of sulfoxides. The structural mechanisms for adverse stereoselectivity between LnPAMO-Mu15 and TtPAMO towards OPS and the overoxidation of OPS by TtPAMO were revealed, based on which, TtPAMO was rationally designed focused on the flexibility of loops near catalytic sites. The variant TtPAMO-S482Y was screened out with lowest overoxidation degree towards OPS and RPS due to the decreased flexibility of catalytic center than TtPAMO. The success in this study not only proved the rationality of the overoxidation mechanism proposed in this study but also provided hints for the development of BVMOs towards thioether substrate for corresponding sulfoxide preparation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Multi-analyte detection and imaging of extracellular chemical signaling molecules are crucial for understanding brain function and molecular pathology. In this work, we present a 6-μm-pitch, CMOS-based multichemical image sensor that enables the simultaneous visualization and spatiotemporal multimodal analysis of the lactate and proton (H + ) dynamics without any labeling. Using semiconductor lithography, gold electrode patterns functioning as lactate-sensing regions were formed on a potentiometric sensor array. Lactate is detected potentiometrically because of redox reactions using lactate oxidase and horseradish peroxidase. The resulting multichemical image sensor exhibited a pH sensitivity of 65 mV and a superior detection limit of 1 μM for lactate with a reasonable selectivity. Furthermore, diffusion images of lactate and H + distributions were obtained concurrently, allowing for simultaneous real-time imaging of the two chemicals with subcellular resolution. We believe that our novel imaging device can be successfully applied to extracellular microenvironments in tissue or cell samples as an effective bioimaging tool., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:To the best of our knowledge, the named authors have no conflict of interest, financial or otherwise., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Cellulose-binding domains (CBDs) play a vital role in cellulose degradation by enzymes. Despite the strong ability of brown-rot fungi to degrade cellulose in wood, they have been considered to lack or have a low number of enzymes with CBD. Here, we report the C-terminal domain of a lytic polysaccharide monooxygenase from the brown-rot fungus Gloeophyllum trabeum (GtLPMO9A-2) functions as a CBD, classified as a new family of carbohydrate-binding module, CBM104. The amino acid sequence of GtCBM104 shows no similarity to any known CBDs. A BLAST search identified 84 homologous sequences at the C-terminus of some CAZymes, mainly LPMO9, in basidiomycetous genomes. Binding experiments revealed GtCBM104 binds selectively to native crystalline cellulose (cellulose I), but not to artificially modified crystalline or amorphous cellulose, while the typical fungal CBD (CBM1) bound to all cellulosic materials tested. The adsorption efficiency of GtCBM104 to cellulose I was >20-times higher than that of CBM1. Adsorption tests and microscopic observations strongly suggested that GtCBM104 binds to the hydrophilic regions of cellulose microfibrils, while CBM1 recognizes the hydrophobic surface. The discovery of GtCBM104 strongly suggests that the contribution of CBD to the cellulose enzymatic degradation mechanism of brown-rot fungi is much larger than previously thought., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Antibody-enzyme complexes (AECs) are ideal for immunosensing. Although AECs using antibody fragments can be produced by bacterial hosts, their low affinity limits their sensing applications. We have improved the affinity of AECs by combining two antibodies using Catcher/Tag systems; however, it requires multiple antibodies and an enzyme production process. In this study, to realize the production of AECs harboring multiple antibody fragments in a single production process, we report a versatile development method of unique AECs based on a multimeric enzyme structure. Using the homotetrameric enzyme, lactate oxidase (LOx), as a labeling enzyme, tetravalent AECs were developed as an electrochemical immunosensor. Homogeneous tetravalent AECs were successfully fabricated by fusing the anti-epidermal growth factor receptor (EGFR) variable domain of a heavy chain of heavy chain antibody to the N-terminus of LOx. The prepared AECs bound to EGFR, maintain their enzyme activity, and worked well as sensing elements in electrochemical sandwich enzyme-linked immunosorbent assay. Moreover, tetravalent AECs exhibited higher sensitivity than monovalent AECs because of their avidity. The fabricated AECs were successfully used in a wash-free homogeneous electrochemical detection system combined with magnetic separation. Our findings offer new insights into the applications of the LOx tetrameric enzyme for the fabrication of AECs with tetravalent antibodies, which may serve as scaffolds for immunosensors., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

2-ketoglutarate (2-KG)/Fe 2+ -dependent dioxygenases can catalyze the highly specific regio- and stereoselective functionalization of C(sp 3 )-H bond of complex compounds under mild reaction conditions. Hyoscyamine 6β-hydroxylase (H6H), a member of these dioxygenases, catalyzes two consecutive oxidation reactions in the synthesis of scopolamine. The first reaction is the hydroxylation of hyoscyamine to 6β-hydroxyhyoscyamine and the second is epoxidation of 6β-hydroxyhyoscyamine. This paper introduces the catalytic mechanism, substrate scope, and application of H6H and evaluates the possibility of this enzyme as a biocatalyst for the functionalization of C(sp 3 )-H bond in complex compounds with different structural characteristics via hydroxylation or epoxidation, providing a theoretical basis for modification and application of this enzyme.

Aspartyl/asparaginyl hydroxylase (AspH) catalyzes the post-translational hydroxylations of vital human proteins, playing an essential role in maintaining their biological functions. Single-point mutations in the Second Coordination Sphere (SCS) and long-range (LR) residues of AspH have been linked to pathological conditions such as the ophthalmologic condition Traboulsi syndrome and chronic kidney disease (CKD). Although the clinical impacts of these mutations are established, there is a critical knowledge gap regarding their specific atomistic effects on the catalytic mechanism of AspH. In this study, we report integrated computational investigations on the potential mechanistic implications of four mutant forms of human AspH with clinical importance: R735W, R735Q, R688Q, and G434V. All the mutant forms exhibited altered binding interactions with the co-substrate 2-oxoglutarate (2OG) and the main substrate in the ferric-superoxo and ferryl complexes, which are critical for catalysis, compared to the wild-type (WT). Importantly, the mutations strongly influence the energetics of the frontier molecular orbitals (FMOs) and, thereby, the activation energies for the hydrogen atom transfer (HAT) step compared to the WT AspH. Insights from our study can contribute to enzyme engineering and the development of selective modulators for WT and mutants of AspH, ultimately aiding in treating cancers, Traboulsi syndrome and, CKD., (© 2024 Wiley-VCH GmbH.)

Baeyer-Villiger monooxygenases (BVMOs) are NAD(P)H-dependent flavoproteins that convert ketones to esters and lactones. While these enzymes offer an appealing alternative to traditional Baeyer-Villiger oxidations, these proteins tend to be either too unstable or exhibit too narrow of a substrate scope for implementation as industrial biocatalysts. Here, sequence similarity networks were used to search for novel BVMOs that are both stable and promiscuous. Our genome mining led to the identification of an enzyme from Chloroflexota bacterium (strain G233) dubbed ssnBVMO that exhibits i) the highest melting temperature of any naturally sourced BVMO (62.5 °C), ii) a remarkable kinetic stability across a wide range of conditions, similar to those of PAMO and PockeMO, iii) optimal catalysis at 50 °C, and iv) a broad substrate scope that includes linear aliphatic, aromatic, and sterically bulky ketones. Subsequent quantitative assays using propiophenone demonstrated >95 % conversion. Several fusions were also constructed that linked ssnBVMO to a thermostable phosphite dehydrogenase. These fusions can recycle NADPH and catalyze oxidations with sub-stoichiometric quantities of this expensive cofactor. Characterization of these fusions permitted identification of PTDH-L1-ssnBVMO as the most promising protein that could have utility as a seed sequence for enzyme engineering campaigns aiming to develop biocatalysts for Baeyer-Villiger oxidations., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Cytochrome P450 monooxygenases (P450s) are well recognized as versatile bio-oxidation catalysts. However, the catalytic functions of P450s are highly dependent on NAD(P)H and redox partner proteins. Our group has recently reported the use of a dual-functional small molecule (DFSM) for generating peroxygenase activity of P450BM3, a long-chain fatty acid hydroxylase from Bacillus megaterium . The DFSM-facilitated P450BM3 peroxygenase system exhibited excellent peroxygenation activity and regio-/enantioselectivity for various organic substrates, such as styrenes, thioanisole, small alkanes, and alkylbenzenes. Very recently, we demonstrated that the DFSM-facilitated P450BM3 peroxygenase could be switched to a peroxidase by engineering the redox-sensitive tyrosine residues in P450BM3. Given the great potential of P450 peroxidase for C-H oxyfunctionalization, we herein report scrutiny of the effect of mutating redox-sensitive residues on peroxidase activity by deeply screening all redox-sensitive residues of P450BM3, namely methionines, tryptophans, cysteines, and phenylalanines. As a result, six beneficial mutations at positions M212, F81, M112, F173, M177, and F77 were screened out from 78 constructed mutants, and significantly enhanced the peroxidase activity of P450BM3 in the presence of Im-C6-Phe, a typical DFSM molecule. Further combination of the beneficial mutations resulted in a more than 100-fold improvement in peroxidase activity compared with that of the combined parent enzyme and DFSM, comparable to or better than most natural peroxidases. In addition, mutations of redox-sensitive residues even dramatically increased, by more than 300-fold, the peroxidase activity of the starting F87A enzyme in the absence of the DFSM, despite the far lower apparent catalytic turnover number compared with the DFSM-P450 system. This study provides new insights and a potential strategy for regulating the catalytic promiscuity of P450 enzymes for multiple functional oxidations.

Hyoscyamine 6β-hydroxylase (H6H) is an iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase that produces the prolifically administered antinausea drug, scopolamine. After its namesake hydroxylation reaction, H6H then couples the newly installed C6 oxygen to C7 to produce the drug's epoxide functionality. Oxoiron(IV) (ferryl) intermediates initiate both reactions by cleaving C-H bonds, but it remains unclear how the enzyme switches the target site and promotes (C6)O-C7 coupling in preference to C7 hydroxylation in the second step. In one possible epoxidation mechanism, the C6 oxygen would─analogously to mechanisms proposed for the Fe/2OG halogenases and, in our more recent study, N -acetylnorloline synthase (LolO)─coordinate as alkoxide to the C7-H-cleaving ferryl intermediate to enable alkoxyl coupling to the ensuing C7 radical. Here, we provide structural and kinetic evidence that H6H does not employ substrate coordination or repositioning for the epoxidation step but instead exploits the distinct spatial dependencies of competitive C-H cleavage (C6 vs C7) and C-O-coupling (oxygen rebound vs cyclization) steps to promote the two-step sequence. Structural comparisons of ferryl-mimicking vanadyl complexes of wild-type H6H and a variant that preferentially 7-hydroxylates instead of epoxidizing 6β-hydroxyhyoscyamine suggest that a modest (∼10°) shift in the Fe-O-H(C7) approach angle is sufficient to change the outcome. The 7-hydroxylation:epoxidation partition ratios of both proteins increase more than 5-fold in 2 H 2 O, reflecting an epoxidation-specific requirement for cleavage of the alcohol O-H bond, which, unlike in the LolO oxacyclization, is not accomplished by iron coordination in advance of C-H cleavage.

Among the enzymes derived from fungus that act on polysaccharides, lytic polysaccharide monooxygenase (LPMOs) has emerged as a new member with complex reaction mechanisms and high efficiency in dealing with recalcitrant crystalline polysaccharides. This study reported the characteristics, structure, and biochemical properties of a novel LPMO from Talaromyces sedimenticola (namely MaLPMO9K) obtained from the Mariana Trench. MaLPMO9K was a multi-domain protein combined with main body and a carbohydrate-binding module. It was heterologously expressed in E. coli for analyzing peroxidase activity in reactions with the substrate 2,6-DMP, where H 2 O 2 serves as a co-substrate. Optimal peroxidase activity for MaLPMO9K was observed at pH 8 and 25 °C, achieving the best V max value of 265.2 U·g -1 . In addition, MaLPMO9K also demonstrated the ability to treat cellulose derivatives, and cellobiose substrates without the presence of reducing agents., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The current study is the first to describe the temporal and differential transcriptional expression of two lytic polysaccharide monooxygenase (LPMO) genes of Rasamsonia emersonii in response to various carbon sources. The mass spectrometry based secretome analysis of carbohydrate active enzymes (CAZymes) expression in response to different carbon sources showed varying levels of LPMOs (AA9), AA3, AA7, catalase, and superoxide dismutase enzymes pointing toward the redox-interplay between the LPMOs and auxiliary enzymes. Moreover, it was observed that cello-oligosaccharides have a negative impact on the expression of LPMOs, which has not been highlighted in previous reports. The LPMO1 (30 kDa) and LPMO2 (47 kDa), cloned and expressed in Pichia pastoris, were catalytically active with (k cat /K m ) of 6.6×10 -2 mg -1 ml min -1 and 1.8×10 -2 mg -1 ml min -1 against Avicel, respectively. The mass spectrometry of hydrolysis products of Avicel/carboxy methyl cellulose (CMC) showed presence of C 1 /C 4 oxidized oligosaccharides indicating them to be Type 3 LPMOs. The 3D structural analysis of LPMO1 and LPMO2 revealed distinct arrangements of conserved catalytic residues at their active site. The developed enzyme cocktails consisting of cellulase from R. emersonii mutant M36 supplemented with recombinant LPMO1/LPMO2 resulted in significantly enhanced saccharification of steam/acid pretreated unwashed rice straw slurry from PRAJ industries (Pune, India). The current work indicates that LPMO1 and LPMO2 are catalytically efficient and have a high degree of thermostability, emphasizing their usefulness in improving benchmark enzyme cocktail performance. KEY POINTS: • Mass spectrometry depicts subtle interactions between LPMOs and auxiliary enzymes. • Cello-oligosaccharides strongly downregulated the LPMO1 expression. • Developed LPMO cocktails showed superior hydrolysis in comparison to CellicCTec3., (© 2024. The Author(s).)

Polyacrylamide (PAM) is a high-molecular-weight polymer with extensive applications. However, the inefficient natural degradation of PAM results in environmental accumulation of the polymer. Biodegradation is an environmentally friendly approach in the field of PAM treatment. The first phase of PAM biodegradation is the deamination of PAM, forming the product poly(acrylic acid) (PAA). The second phase of PAM biodegradation involves the cleavage of PAA into small molecules, which is a crucial step in the degradation pathway of PAM. However, the enzyme that catalyzes the degradation of PAA and the molecular mechanism remain unclear. Here, a novel monooxygenase PCX02514 is identified as the key enzyme for PAA degradation. Through biochemical experiments, the monooxygenase PCX02514 oxidizes PAA with the participation of NADPH, causing the cleavage of carbon chains and a decrease in the molecular weight of PAA. In addition, the crystal structure of the monooxygenase PCX02514 is solved at a resolution of 1.97 Å. The active pocket is in a long cavity that extends from the C-terminus of the TIM barrel to the protein surface and exhibits positive electrostatic potential, thereby causing the migration of oxygen-negative ions into the active pocket and facilitating the reaction between the substrates and monooxygenase PCX02514. Moreover, Arg10-Arg125-Ser186-Arg187-His253 are proposed as potential active sites in monooxygenase PCX02514. Our research characterizes the molecular mechanism of this monooxygenase, providing a theoretical basis and valuable tools for PAM bioremediation.

MICAL proteins represent a unique family of actin regulators crucial for synapse development, membrane trafficking, and cytokinesis. Unlike classical actin regulators, MICALs catalyze the oxidation of specific residues within actin filaments to induce robust filament disassembly. The potent activity of MICALs requires tight control to prevent extensive damage to actin cytoskeleton. However, the molecular mechanism governing MICALs' activity regulation remains elusive. Here, we report the cryo-EM structure of MICAL1 in the autoinhibited state, unveiling a head-to-tail interaction that allosterically blocks enzymatic activity. The structure also reveals the assembly of C-terminal domains via a tripartite interdomain interaction, stabilizing the inhibitory conformation of the RBD. Our structural, biochemical, and cellular analyses elucidate a multi-step mechanism to relieve MICAL1 autoinhibition in response to the dual-binding of two Rab effectors, revealing its intricate activity regulation mechanisms. Furthermore, our mutagenesis study of MICAL3 suggests the conserved autoinhibition and relief mechanisms among MICALs., (© 2024. The Author(s).)

Lytic polysaccharide monooxygenase (LPMO)-catalyzed oxidative processes play a major role in natural biomass conversion. Despite their oxidative cleavage at the surface of polysaccharides, understanding of their mode of action, and the impact of structural patterns of the cellulose fiber on LPMO activity is still not fully understood. In this work, we investigated the action of two different LPMOs from Podospora anserina on celluloses showing different structural patterns. For this purpose, we prepared cellulose II and cellulose III allomorphs from cellulose I cotton linters, as well as amorphous cellulose. LPMO action was monitored in terms of surface morphology, molar mass changes and monosaccharide profile. Both PaLPMO9E and PaLPMO9H were active on the different cellulose allomorphs (I, II and III), and on amorphous cellulose (PASC) whereas they displayed a different behavior, with a higher molar mass decrease observed for cellulose I. Overall, the pretreatment with LPMO enzymes clearly increased the accessibility of all types of cellulose, which was quantified by the higher carboxylate content after carboxymethylation reaction on LPMO-pretreated celluloses. This work gives more insight into the action of LPMOs as a tool for deconstructing lignocellulosic biomass to obtain new bio-based building blocks., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jean-Guy Berrin reports financial support was provided by Carnot 3BCar Institute. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The synthesis of steroids is challenging through multistep steroidal core modifications with high site-selectivity and productivity. In this work, a novel enzymatic cascade system was constructed for synthesis of testolactone by specific C17 lactonization/Δ 1 -dehydrogenation from inexpensive androstenedione using an engineered polycyclic ketone monooxygenase (PockeMO) and an appropriate 3-ketosteroid-Δ 1 -dehydrogenase (ReKstD). The focused saturation mutagenesis in the substrate binding pocket was implemented for evolution of PockeMO to eliminate the bottleneck effect. A best mutant MU3 (I225L/L226V/L532Y) was obtained with 20-fold higher specific activity compared to PockeMO. The catalytic efficiency (k cat /Km) of MU3 was 171-fold higher and the substrate scope shifted to polycyclic ketones. Molecular dynamic simulations suggested that the activity was improved by stabilization of the pre-lactonization state and generation of productive orientation of 4-AD mediated by distal L532Y mutation. Based on that, the three genes, MU3, ReKstD and a ketoreductase for NADPH regeneration, were rationally integrated in one cell via expression fine-tuning to form the efficient single cell catalyst E. coli S9. The single whole-cell biocatalytic process was scaled up and could generate 9.0 g/L testolactone with the high space time yield of 1 g/L/h without steroidal by-product, indicating the potential for site-specific and one-pot synthesis of steroid., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2024. Published by Elsevier B.V.)

The unspecific peroxygenase (UPO) from Agrocybe aegerita (r Aae UPO-PaDa-I-H) is an effective and practical biocatalyst for the oxidative expansion of furfuryl alcohols/amines on a preparative scale, using the Achmatowicz and aza-Achmatowicz reaction. The high activity and stability of the enzyme, which can be produced on a large scale as an air-stable lyophilised powder, renders it a versatile and scalable biocatalyst for the preparation of synthetically valuable 6-hydroxypyranones and dihydropiperidinones. In several cases, the biotransformation out-performed the analogous chemo-catalysed process, and operates under milder and greener reaction conditions.

Flavin-containing monooxygenase from Methylophaga sp. ( m FMO) 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 m FMO 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 ( Ni FMO). 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 m FMO influence selectivity, representing a tunable biocatalyst for enantioselective sulfoxidations.

The high level of lactate in tumor microenvironment not only promotes tumor development and metastasis, but also induces immune escape, which often leads to failures of various tumor therapy strategies. We here report a sono-triggered cascade lactate depletion strategy by using semiconducting polymer nanoreactors (SPN LCu ) for cancer cuproptosis-immunotherapy. The SPN LCu mainly contain a semiconducting polymer as sonosensitizer, lactate oxidase (LOx) conjugated via a reactive oxygen species (ROS)-cleavable linker and chelated Cu 2+ . Upon ultrasound (US) irradiation, the semiconducting polymer generates singlet oxygen ( 1 O 2 ) to cut ROS-cleavable linker to allow the release of LOx that catalyzes lactate depletion to produce hydrogen peroxide (H 2 O 2 ). The Cu 2+ will be reduced to Cu + in tumor microenvironment, which reacts with the produced H 2 O 2 to obtain hydroxyl radical (⋅OH) that further improves LOx release via destroying ROS-cleavable linkers. As such, sono-triggered cascade release of LOx achieves effective lactate depletion, thus relieving immunosuppressive roles of lactate. Moreover, the toxic Cu + induces cuproptosis to cause immunogenic cell death (ICD) for activating antitumor immunological effect. SPN LCu are used to treat both subcutaneous and deep-tissue orthotopic pancreatic cancer with observably enhanced efficacy in restricting the tumor growths. This study thus provides a precise and effective lactate depletion tactic for cancer therapy., (© 2024 Wiley-VCH GmbH.)

Enzymatic oxygenation of various cyclic ketones into lactones via Baeyer-Villiger monooxygenases (BVMOs) could provide a promising route for synthesizing fragrances and pharmaceutical ingredients. However, unsatisfactory catalytic activity and thermostability restricted their applications in the pharmaceutical and food industries. In this study, we successfully improved the catalytic activity and thermostability of a Baeyer-Villiger monooxygenase ( Og BVMO) from Oceanicola granulosus by reshaping the binding pocket. As a result, mutant Og BVMO-Re displayed a 1.0- to 6.4-fold increase in the activity toward branched cyclic ketones tested, accompanied by a 3 °C higher melting point, and a 2-fold longer half-life time ( t 1/2 (45 °C)). Molecular dynamics simulations revealed that reshaping the binding pocket achieved strengthened motion correlation between amino acid residues, appropriate size of the substrate-binding pocket, beneficial surface characteristics, lower energy barriers, and shorter nucleophilic distance. This study well demonstrated the trade-off between the enzyme activity and thermostability by reshaping the substrate-binding pocket, paving the way for further engineering other enzymes.

CreE is a flavin-dependent monooxygenase (FMO) that catalyzes three sequential nitrogen oxidation reactions of L-aspartate to produce nitrosuccinate, contributing to the biosynthesis of the antimicrobial and antiproliferative nautral product, cremeomycin. This compound contains a highly reactive diazo functional group for which the reaction of CreE is essential to its formation. Nitro and diazo functional groups can serve as potent electrophiles, important in some challenging nucleophilic addition reactions. Formation of these reactive groups positions CreE as a promising candidate for biomedical and synthetic applications. Here, we present the catalytic mechanism of CreE and the identification of active site residues critical to binding L-aspartate, aiding in future enzyme engineering efforts. Steady-state analysis demonstrated that CreE is very specific for NADPH over NADH and performs a highly coupled reaction with L-aspartate. Analysis of the rapid-reaction kinetics showed that flavin reduction is very fast, along with the formation of the oxygenating species, the C4a-hydroperoxyflavin. The slowest step observed was the dehydration of the flavin. Structural analysis and site-directed mutagenesis implicated T65, R291, and R440 in the binding L-aspartate. The data presented describes the catalytic mechanism and the active site architecture of this unique FMO., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

A novel optical lactate biosensor is presented that utilizes a colorimetric interaction between H 2 O 2 liberated by a binary enzymatic reaction and bis(neocuproine)copper(II) complex ([Cu(Nc) 2 ] 2+ ) known as CUPRAC (cupric reducing antioxidant capacity) reagent. In the first step, lactate oxidase (LOx) and pyruvate oxidase (POx) were separately immobilized on silanized magnetite nanoparticles (SiO 2 @Fe 3 O 4 NPs), and thus, 2 mol of H 2 O 2 was released per 1 mol of the substrate due to a sequential enzymatic reaction of the mixture of LOx-SiO 2 @Fe 3 O 4 and POx-SiO 2 @Fe 3 O 4 NPs with lactate and pyruvate, respectively. In the second step, the absorbance at 450 nm of the yellow-orange [Cu(Nc) 2 ] + complex formed through the color reaction of enzymatically produced H 2 O 2 with [Cu(Nc) 2 ] 2+ was recorded. The results indicate that the developed colorimetric binary enzymatic biosensor exhibits a broad linear range of response between 0.5 and 50.0 µM for lactate under optimal conditions with a detection limit of 0.17 µM. The fabricated biosensor did not respond to other saccharides, while the positive interferences of certain reducing compounds such as dopamine, ascorbic acid, and uric acid were minimized through their oxidative removal with a pre-oxidant (NaBiO 3 ) before enzymatic and colorimetric reactions. The fabricated optical biosensor was applied to various samples such as artificial blood, artificial/real sweat, and cow milk. The high recovery values (close to 100%) achieved for lactate-spiked samples indicate an acceptable accuracy of this colorimetric biosensor in the determination of lactate in real samples. Due to the increase in H 2 O 2 production with the bienzymatic lactate sensor, the proposed method displays double-fold sensitivity relative to monoenzymatic biosensors and involves a neat color reaction with cupric-neocuproine having a clear stoichiometry as opposed to the rather indefinite stoichiometry of analogous redox dye methods., (© 2024. The Author(s).)

Baeyer-Villiger monooxygenases belong to a family of flavin-binding proteins that catalyze the Baeyer-Villiger (BV) oxidation of ketones to produce lactones or esters, which are important intermediates in pharmaceuticals or sustainable materials. Phenylacetone monooxygenase (PAMO) from Thermobifida fusca with moderate thermostability catalyzes the oxidation of aryl ketone substrates, but is limited by high specificity and narrow substrate scope. In the present study, we applied loop optimization by loop swapping followed by focused saturation mutagenesis in order to evolve PAMO mutants capable of catalyzing the regioselective BV oxidation of cyclohexanone and cyclobutanone derivatives with formation of either normal or abnormal esters or lactones. We further modulated PAMO to increase enantioselectivity. Crystal structure studies indicate that rotation occurs in the NADP-binding domain and that the high B-factor region is predominantly distributed in the catalytic pocket residues. Computational analyses further revealed dynamic character in the catalytic pocket and reshaped hydrogen bond interaction networks, which is more favorable for substrate binding. Our study provides useful insights for studying enzyme-substrate adaptations., (© 2024 Wiley-VCH GmbH.)

Intrinsic characteristics of microorganisms, including non-specific metabolism sites, toxic byproducts, and uncontrolled proliferation constrain their exploitation in medical applications such as tumor therapy. Here, the authors report an engineered biohybrid that can efficiently target cancerous sites through a pre-determined metabolic pathway to enable precise tumor ablation. In this system, DH5α Escherichia coli is engineered by the introduction of hypoxia-inducible promoters and lactate oxidase genes, and further surface-armored with iron-doped ZIF-8 nanoparticles. This bioengineered E. coli can produce and secrete lactate oxidase to reduce lactate concentration in response to hypoxic tumor microenvironment, as well as triggering immune activation. The peroxidase-like functionality of the nanoparticles extends the end product of the lactate metabolism, enabling the conversion of hydrogen peroxide (H 2 O 2 ) into highly cytotoxic hydroxyl radicals. This, coupled with the transformation of tirapazamine loaded on nanoparticles to toxic benzotriazinyl, culminates in severe tumor cell ferroptosis. Intravenous injection of this biohybrid significantly inhibits tumor growth and metastasis., (© 2024 Wiley‐VCH GmbH.)

Background: White-rot fungi and bacteria communities are unique ecosystems with different types of symbiotic interactions occurring during wood decomposition, such as cooperation, mutualism, nutritional competition, and antagonism. The role of chitin-active lytic polysaccharide monooxygenases (LPMOs) in these symbiotic interactions is the subject of this study., Method: In this study, bioinformatics tools were used to analyze the sequence and structure of putative LPMOs mined by hidden Markov model (HMM) profiles from the bacterial metagenomic DNA database of collected humus samples around white-rot fungi in Cuc Phuong primary forest, Vietnam. Two genes encoding putative LPMOs were expressed in E. coli and purified for enzyme activity assay., Result: Thirty-one full-length proteins annotated as putative LPMOs according to HMM profiles were confirmed by amino acid sequence comparison. The comparison results showed that although the amino acid sequences of the proteins were very different, they shared nine conserved amino acids, including two histidine and one phenylalanine that characterize the H1-Hx-Yz motif of the active site of bacterial LPMOs. Structural analysis of these proteins revealed that they are multidomain proteins with different functions. Prediction of the catalytic domain 3-D structure of these putative LPMOs using Alphafold2 showed that their spatial structures were very similar in shape, although their protein sequences were very different. The results of testing the activity of proteins GL0247266 and GL0183513 show that they are chitin-active LPMOs. Prediction of the 3-D structures of these two LPMOs using Alphafold2 showed that GL0247266 had five functional domains, while GL0183513 had four functional domains, two of which that were similar to the GbpA_2 and GbpA_3 domains of protein GbpA of Vibrio cholerae bacteria. The GbpA_2 - GbpA_3 complex was also detected in 11 other proteins. Based on the structural characteristics of functional domains, it is possible to hypothesize the role of chitin-active GbpA-like LPMOs in the relationship between fungal and bacterial communities coexisting on decomposing trees in primary forests., Competing Interests: The authors declare there are no competing interests, (©2024 Truong et al.)

In this paper, we employed a multidisciplinary approach, combining experimental techniques and density functional theory (DFT) calculations to elucidate key features of the copper coordination environment of the bacterial lytic polysaccharide monooxygenase (LPMO) from Serratia marcescens ( Sm AA10). The structure of the holo -enzyme was successfully obtained by X-ray crystallography. We then determined the copper(II) binding affinity using competing ligands and observed that the affinity of the histidine brace ligands for copper is significantly higher than previously described. UV-vis, advanced electron paramagnetic resonance (EPR), and X-ray absorption spectroscopy (XAS) techniques, including high-energy resolution fluorescence detected (HERFD) XAS, were further used to gain insight into the copper environment in both the Cu(II) and Cu(I) redox states. The experimental data were successfully rationalized by DFT models, offering valuable information on the electronic structure and coordination geometry of the copper center. Finally, the Cu(II)/Cu(I) redox potential was determined using two different methods at ca . 350 mV vs NHE and rationalized by DFT calculations. This integrated approach not only advances our knowledge of the active site properties of Sm AA10 but also establishes a robust framework for future studies of similar enzymatic systems.

Baeyer-Villiger monooxygenases (BVMOs) play crucial roles in the core-structure modification of natural products. They catalyze lactone formation by selective oxygen insertion into a carbon-carbon bond adjacent to a carbonyl group (Baeyer-Villiger oxidation, BVO). The homologous bacterial BVMOs, BraC and PxaB, thereby process bicyclic dihydroindolizinone substrates originating from a bimodular nonribosomal peptide synthetase (BraB or PxaA). While both enzymes initially catalyze the formation of oxazepine-dione intermediates following the identical mechanism, the final natural product spectrum diverges. For the pathway involving BraC, the exclusive formation of lipocyclocarbamates, the brabantamides, was reported. The pathway utilizing PxaB solely produces pyrrolizidine alkaloids, the pyrrolizixenamides. Surprisingly, replacing pxaB within the pyrrolizixenamide biosynthetic pathway by braC does not change the product spectrum to brabantamides. Factors controlling this product selectivity have remained elusive. In this study, we set out to solve this puzzle by combining the total synthesis of crucial pathway intermediates and anticipated products with in-depth functional in vitro studies on both recombinant BVMOs. This work shows that the joint oxazepine-dione intermediate initially formed by both BVMOs leads to pyrrolizixenamides upon nonenzymatic hydrolysis, decarboxylative ring contraction, and dehydration. Brabantamide biosynthesis is enzyme-controlled, with BraC efficiently transforming all the accepted substrates into its cognate final product scaffold. PxaB, in contrast, shows only considerable activity toward brabantamide formation for the substrate analog with a natural brabantamide-type side chain structure, revealing substrate-controlled product selectivity.

Indole monooxygenases (IMOs) are enzymes from the family of Group E monooxygenases, requiring flavin adenine dinucleotide (FAD) for their activities. IMOs play important roles in both sulfoxidation and epoxidation reactions. The broad substrate range and high selectivity of IMOs make them promising biocatalytic tools for synthesizing chiral compounds. In the present study, quantum chemical calculations using the cluster approach were performed to investigate the reaction mechanism and the enantioselectivity of the IMO from Variovorax paradoxus EPS ( Vp IndA1). The sulfoxidation of methyl phenyl sulfide (MPS) and the epoxidation of indene were chosen as the representative reactions. The calculations confirmed that the FAD OOH intermediate is the catalytic species in the Vp IndA1 reactions. The oxidation of MPS adopts a one-step mechanism involving the direct oxygen-transfer from FAD OOH to the substrate and the proton transfer from the -OH group back to FAD, while the oxidation of indene follows a stepwise mechanism involving a carbocation intermediate. It was computationally predicted that Vp IndA1 prefers the formation of ( S )-product for the MPS sulfoxidation and (1 S ,2 R )-product for the indene epoxidation, consistent with the experimental observations. Importantly, the factors controlling the stereo-preference of the two reactions are identified. The findings in the present study provide valuable insights into the Vp IndA1-catalyzed reactions, which are essential for the rational design of this enzyme and other IMOs for industrial applications. It is also worth emphasizing that the quantum chemical cluster approach is again demonstrated to be powerful in studying the enantioselectivity of enzymatic reactions.

Enzymatic electrophilic halogenation is a mild tool for functionalization of diverse organic compounds. Only a few groups of native halogenases are capable of catalyzing such a reaction. In this study, we used a mechanism-guided strategy to discover the electrophilic halogenation activity catalyzed by non-native halogenases. As the ability to form a hypohalous acid (HOX) is key for halogenation, flavin-dependent monooxygenases/oxidases capable of forming C4a-hydroperoxyflavin (Fl C4a-OOH ), such as dehalogenase, hydroxylases, luciferase and pyranose-2-oxidase (P2O), and flavin reductase capable of forming H 2 O 2 were explored for their abilities to generate HOX in situ. Transient kinetic analyses using stopped-flow spectrophotometry/fluorometry and product analysis indicate that Fl C4a-OOH in dehalogenases, selected hydroxylases and luciferases, but not in P2O can form HOX; however, the HOX generated from Fl C4a-OOH cannot halogenate their substrates. Remarkably, in situ H 2 O 2 generated by P2O can form HOI and also iodinate various compounds. Because not all enzymes capable of forming Fl C4a-OOH can react with halides to form HOX, QM/MM calculations, site-directed mutagenesis and structural analysis were carried out to elucidate the mechanism underlying HOX formation and characterize the active site environment. Our findings shed light on identifying new halogenase scaffolds besides the currently known enzymes and have invoked a new mode of chemoenzymatic halogenation., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

OxaD is a flavin-dependent monooxygenase (FMO) responsible for catalyzing the oxidation of an indole nitrogen atom, resulting in the formation of a nitrone. Nitrones serve as versatile intermediates in complex syntheses, including challenging reactions like cycloadditions. Traditional organic synthesis methods often yield limited results and involve environmentally harmful chemicals. Therefore, the enzymatic synthesis of nitrone-containing compounds holds promise for more sustainable industrial processes. In this study, we explored the catalytic mechanism of OxaD using a combination of steady-state and rapid-reaction kinetics, site-directed mutagenesis, spectroscopy, and structural modeling. Our investigations showed that OxaD catalyzes two oxidations of the indole nitrogen of roquefortine C, ultimately yielding roquefortine L. The reductive-half reaction analysis indicated that OxaD rapidly undergoes reduction and follows a "cautious" flavin reduction mechanism by requiring substrate binding before reduction can take place. This characteristic places OxaD in class A of the FMO family, a classification supported by a structural model featuring a single Rossmann nucleotide binding domain and a glutathione reductase fold. Furthermore, our spectroscopic analysis unveiled both enzyme-substrate and enzyme-intermediate complexes. Our analysis of the oxidative-half reaction suggests that the flavin dehydration step is the slow step in the catalytic cycle. Finally, through mutagenesis of the conserved D63 residue, we demonstrated its role in flavin motion and product oxygenation. Based on our findings, we propose a catalytic mechanism for OxaD and provide insights into the active site architecture within class A FMOs.

Flavoenzymes catalyze numerous redox reactions including the transfer of an O 2 -derived oxygen atom to organic substrates, while the other one is reduced to water. Investigation of some of these monooxygenases led to a detailed understanding of their catalytic cycle, which involves the flavin-C 4α -(hydro)peroxide as hallmark oxygenating species, and newly discovered flavoprotein monooxygenases were generally assumed to operate similarly. However, discoveries in recent years revealed a broader mechanistic versatility, including enzymes that utilize flavin-N 5 oxygen adducts for catalysis in the form of the flavin-N 5 -(hydro)peroxide and the flavin-N 5 -oxide species. In this review, I will highlight recent developments in that area, including noncanonical flavoenzymes from natural product biosynthesis and sulfur metabolism that provide first insights into the chemical properties of these species. Remarkably, some enzymes may even combine the flavin-N 5 -peroxide and the flavin-N 5 -oxide species for consecutive oxygen-transfers to the same substrate and thereby in essence operate as dioxygenases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Enzymes are crucial for the emergence and sustenance of life on earth. How they became catalytically active during their evolution is still an open question. Two opposite explanations are plausible: acquiring a mechanism in a series of discrete steps or all at once in a single evolutionary event. Here, we use molecular phylogeny, ancestral sequence reconstruction, and biochemical characterization to follow the evolution of a specialized group of flavoprotein monooxygenases, the bacterial Baeyer-Villiger monooxygenases (BVMOs). These enzymes catalyze an intricate chemical reaction relying on three different elements: a reduced nicotinamide cofactor, dioxygen, and a substrate. Characterization of ancestral BVMOs shows that the catalytic mechanism evolved in a series of steps starting from a FAD-binding protein and further acquiring reactivity and specificity toward each of the elements participating in the reaction. Together, the results of our work portray how an intrinsically complex catalytic mechanism emerged during evolution., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Due to its detrimental impact on human health and the environment, regulations demand ultralow sulfur levels on fossil fuels, in particular in diesel. However, current desulfurization techniques are expensive and cannot efficiently remove heteroaromatic sulfur compounds, which are abundant in crude oil and concentrate in the diesel fraction after distillation. Biodesulfurization via the four enzymes of the metabolic 4S pathway of the bacterium Rhodococcus erythropolis (DszA-D) is a possible solution. However, the 4S pathway needs to operate at least 500 times faster for industrial applicability, a goal currently pursued through enzyme engineering. In this work, we unveil the catalytic mechanism of the flavin monooxygenase DszA. Surprisingly, we found that this enzyme follows a recently proposed atypical mechanism that passes through the formation of an N 5 OOH intermediate at the re side of the cofactor, aided by a well-defined, predominantly hydrophobic O 2 pocket. Besides clarifying the unusual chemical mechanism of the complex DszA enzyme, with obvious implications for understanding the puzzling chemistry of flavin-mediated catalysis, the result is crucial for the rational engineering of DszA, contributing to making biodesulfurization attractive for the oil refining industry.

In this paper, a novel fluorescent detection method for glucose and lactic acid was developed based on fluorescent iron nanoclusters (Fe NCs). The Fe NCs prepared using hemin as the main raw material exhibited excellent water solubility, bright red fluorescence, and super sensitive response to hydrogen peroxide (H 2 O 2 ). This paper demonstrates that Fe NCs exhibit excellent peroxide-like activity, catalyzing H 2 O 2 to produce hydroxyl radicals ( • OH) that can quench the red fluorescence of Fe NCs. In this paper, a new type of glucose sensor was established by combining Fe NCs with glucose oxidase (GluOx). With the increase in glucose content, the fluorescence of Fe NCs decreases correspondingly, and the glucose content can be detected in the scope of 0-200 μmol·L -1 (μM). Similarly, the lactic acid sensor can also be established by combining Fe NCs with lactate oxidase (LacOx). With the increase in lactic acid concentration, the fluorescence of Fe NCs decreases correspondingly, and the lactic acid content can be detected in the range of 0-100 μM. Furthermore, Fe NCs were used in the preparation of gel test strip, which can be used to detect H 2 O 2 , glucose and lactic acid successfully by the changes of fluorescent intensity.

Dopamine (DA) signaling is critically important in striatal function, and this metabolically demanding process is fueled largely by glucose. However, DA and glucose are typically studied independently and, as such, the precise relationship between DA release and glucose availability remains unclear. Fast-scan cyclic voltammetry (FSCV) is commonly coupled with carbon-fiber microelectrodes to study DA transients. These microelectrodes can be modified with glucose oxidase (GOx) to generate microbiosensors capable of simultaneously quantifying real-time and physiologically relevant fluctuations of glucose, a nonelectrochemically active substrate, and DA, which is readily oxidized and reduced at the electrode surface. A chitosan hydrogel can be electrodeposited to entrap the oxidase enzyme on the sensor surface for stable, sensitive, and selective codetection of glucose and DA using FSCV. This strategy can also be used to entrap lactate oxidase on the carbon-fiber surface for codetection of lactate and DA. However, these custom probes are individually fabricated by hand, and performance is variable. This study characterizes the physical nature of the hydrogel and its effects on the acquired electrochemical data in the detection of glucose (2.6 mM) and DA (1 μM). The results demonstrate that the electrodeposition of the hydrogel membrane is improved using a linear potential sweep rather than a direct step to the target potential. Electrochemical impedance spectroscopy data relate information on the physical nature of the electrode/solution interface to the electrochemical performance of bare and enzyme-modified carbon-fiber microelectrodes. The electrodeposition waveform and scan rate were characterized for optimal membrane formation and performance. Finally, codetection of both DA/glucose and DA/lactate was demonstrated in intact rat striatum using probes fabricated according to the optimized protocol. Overall, this work improves the reliable fabrication of carbon-fiber microbiosensors for codetection of DA and important energetic substrates that are locally delivered to the recording site to meet metabolic demand.

Oxidoreductases have evolved tyrosine/tryptophan pathways that channel highly oxidizing holes away from the active site to avoid damage. Here we dissect such a pathway in a bacterial LPMO, member of a widespread family of C-H bond activating enzymes with outstanding industrial potential. We show that a strictly conserved tryptophan is critical for radical formation and hole transference and that holes traverse the protein to reach a tyrosine-histidine pair in the protein's surface. Real-time monitoring of radical formation reveals a clear correlation between the efficiency of hole transference and enzyme performance under oxidative stress. Residues involved in this pathway vary considerably between natural LPMOs, which could reflect adaptation to different ecological niches. Importantly, we show that enzyme activity is increased in a variant with slower radical transference, providing experimental evidence for a previously postulated trade-off between activity and redox robustness., (© 2024. The Author(s).)