Engineering the substrate specificity and regioselectivity of Burkholderia thailandensis lipoxygenase.

Lipoxygenases (LOXs) catalyze the regioselective dioxygenation of polyunsaturated fatty acids (PUFAs), generating fatty acid hydroperoxides (FAHPs) with diverse industrial applications. Bacterial LOXs have garnered significant attention in recent years due to their broad activity towards PUFAs, yet knowledge about the structural factors influencing their substrate preferences remains limited. Here, we characterized a bacterial LOX from Burkholderia thailandensis (Bt-LOX), and identified key residues affecting its substrate preference and regioselectivity through site-directed mutagenesis.

Polyphenol Oxidase Activity on Guaiacyl and Syringyl Lignin Units.

The natural heterogeneity of guaiacyl (G) and syringyl (S) compounds resulting from lignin processing hampers their direct use as plant-based chemicals and materials. Herein, we explore six short polyphenol oxidases (PPOs) from lignocellulose-degrading ascomycetes for their capacity to react with G-type and S-type phenolic compounds. All six PPOs catalyze the ortho-hydroxylation of G-type compounds (guaiacol, vanillic acid, and ferulic acid), forming the corresponding methoxy-ortho-diphenols.

Prenylation of aromatic amino acids and plant phenolics by an aromatic prenyltransferase from Rasamsonia emersonii.

Dimethylallyl tryptophan synthases (DMATSs) are aromatic prenyltransferases that catalyze the transfer of a prenyl moiety from a donor to an aromatic acceptor during the biosynthesis of microbial secondary metabolites. Due to their broad substrate scope, DMATSs are anticipated as biotechnological tools for producing bioactive prenylated aromatic compounds. Our study explored the substrate scope and product profile of a recombinant RePT, a novel DMATS from the thermophilic fungus Rasamsonia emersonii.

Versatile ferrous oxidation-xylenol orange assay for high-throughput screening of lipoxygenase activity.

Lipoxygenases (LOXs) catalyze dioxygenation of polyunsaturated fatty acids (PUFAs) into fatty acid hydroperoxides (FAHPs), which can be further transformed into a number of value-added compounds. LOXs have garnered interest as biocatalysts for various industrial applications. Therefore, a high-throughput LOX activity assay is essential to evaluate their performance under different conditions.

The secretome of : Temporal dynamics of plant polysaccharides and lignin degradation.

Despite substantial lignocellulose conversion during mycelial growth, previous transcriptome and proteome studies have not yet revealed how secretomes from the edible mushroom develop and whether they modify lignin models . To clarify these aspects, secretomes collected throughout a 15-day industrial substrate production and from axenic lab-cultures were subjected to proteomics, and tested on polysaccharides and lignin models. Secretomes (day 6-15) comprised endo-acting and substituent-removing glycoside hydrolases, whereas β-xylosidase and glucosidase activities gradually decreased.

Polyphenol Oxidase Products Are Priming Agents for LPMO Peroxygenase Activity.

Polyphenol oxidases catalyze the hydroxylation of monophenols to diphenols, which are reducing agents for lytic polysaccharide monooxygenases (LPMOs) in their degradation of cellulose. In particular, the polyphenol oxidase MtPPO7 from Myceliophthora thermophila converts lignocellulose-derived monophenols, and under the new perspective of the peroxygenase reaction catalyzed by LPMOs, we aim to differentiate the role of the catalytic products of MtPPO7 in priming and fueling of LPMO activity. Exemplified by the activity of MtPPO7 towards guaiacol and by using the benchmark LPMO NcAA9C from Neurospora crassa we show that MtPPO7 catalytic products provide the initial electron for the reduction of Cu(II) to Cu(I) but cannot provide the required reducing power for continuous fueling of the LPMO.

A Clickable Photosystem I, Ferredoxin, and Ferredoxin NADP Reductase Fusion System for Light-Driven NADPH Regeneration.

Photosynthetic organisms like plants, algae, and cyanobacteria use light for the regeneration of dihydronicotinamide dinucleotide phosphate (NADPH). The process starts with the light-driven oxidation of water by photosystem II (PSII) and the released electrons are transferred via the cytochrome b f complex towards photosystem I (PSI). This membrane protein complex is responsible for the light-driven reduction of the soluble electron mediator ferredoxin (Fd), which passes the electrons to ferredoxin NADP reductase (FNR).

AA16 Oxidoreductases Boost Cellulose-Active AA9 Lytic Polysaccharide Monooxygenases from .

Copper-dependent lytic polysaccharide monooxygenases (LPMOs) classified in Auxiliary Activity (AA) families are considered indispensable as synergistic partners for cellulolytic enzymes to saccharify recalcitrant lignocellulosic plant biomass. In this study, we characterized two fungal oxidoreductases from the new AA16 family. We found that AA16A from and AA16A from did not catalyze the oxidative cleavage of oligo- and polysaccharides.

Bacterial lipoxygenases: Biochemical characteristics, molecular structure and potential applications.

Lipoxygenases (LOXs) are enzymes that catalyze dioxygenation of polyunsaturated fatty acids into fatty acid hydroperoxides. The formed fatty acid hydroperoxides are of interest as they can readily be transformed to a number of value-added compounds. LOXs are widely distributed in both eukaryotic and prokaryotic organisms, including humans, animals, plants, fungi and bacteria.

Extending the diversity of Myceliophthora thermophila LPMOs: Two different xyloglucan cleavage profiles.

Lytic polysaccharide monooxygenases (LPMOs) play a key role in enzymatic conversion of plant cell wall polysaccharides. Continuous discovery and functional characterization of LPMOs highly contribute to the tailor-made design and improvement of hydrolytic-activity based enzyme cocktails. In this context, a new MtLPMO9F was characterized for its substrate (xyloglucan) specificity, and MtLPMO9H was further delineated.

Regioselective C4 and C6 Double Oxidation of Cellulose by Lytic Polysaccharide Monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) play a key role in enzymatic degradation of hard-to-convert polysaccharides, such as chitin and cellulose. It is widely accepted that LPMOs catalyze a single regioselective oxidation of the C1 or C4 carbon of a glycosidic linkage, after which the destabilized linkage breaks. Here, a series of novel C4/C6 double oxidized cello-oligosaccharides was discovered.

Oxidized Product Profiles of AA9 Lytic Polysaccharide Monooxygenases Depend on the Type of Cellulose.

Lytic polysaccharide monooxygenases (LPMOs) are essential for enzymatic conversion of lignocellulose-rich biomass in the context of biofuels and platform chemicals production. Considerable insight into the mode of action of LPMOs has been obtained, but research on the cellulose specificity of these enzymes is still limited. Hence, we studied the product profiles of four fungal Auxiliary Activity family 9 (AA9) LPMOs during their oxidative cleavage of three types of cellulose: bacterial cellulose (BC), Avicel PH-101 (AVI), and regenerated amorphous cellulose (RAC).

The Endophytic Fungus Influences Growth of the Nonnatural Host Plant .

is a fungal endophyte from , a perennial plant from the northern part of Asia. Here, we demonstrated an interaction of with , Chinese cabbage, rapeseed, tomato, maize, or sunflower resulting in different phenotypes such as shorter main roots, massive lateral root growth, higher leaf and root biomass, and increased anthocyanin levels. In a variety of cocultivation assays, it was shown that these altered phenotypes are caused by fungal CO, volatile organic compounds, and soluble compounds, notably astins.

Flavoprotein monooxygenases: Versatile biocatalysts.

Flavoprotein monooxygenases (FPMOs) are single- or two-component enzymes that catalyze a diverse set of chemo-, regio- and enantioselective oxyfunctionalization reactions. In this review, we describe how FPMOs have evolved from model enzymes in mechanistic flavoprotein research to biotechnologically relevant catalysts that can be applied for the sustainable production of valuable chemicals. After a historical account of the development of the FPMO field, we explain the FPMO classification system, which is primarily based on protein structural properties and electron donor specificities.

Vanillyl alcohol oxidase.

This review presents a historical outline of the research on vanillyl alcohol oxidase (VAO) from Penicillium simplicissimum, one of the canonical members of the VAO/PCMH flavoprotein family. After describing its discovery and initial biochemical characterization, we discuss the physiological role, substrate scope, and catalytic mechanism of VAO, and review its three-dimensional structure and mechanism of covalent flavinylation. We also explain how protein engineering provided a deeper insight into the role of certain amino acid residues in determining the substrate specificity and enantioselectivity of the enzyme.

Substrate binding tunes the reactivity of hispidin 3-hydroxylase, a flavoprotein monooxygenase involved in fungal bioluminescence.

Fungal bioluminescence was recently shown to depend on a unique oxygen-dependent system of several enzymes. However, the identities of the enzymes did not reveal the full biochemical details of this process, as the enzymes do not bear resemblance to those of other luminescence systems, and thus the properties of the enzymes involved in this fascinating process are still unknown. Here, we describe the characterization of the penultimate enzyme in the pathway, hispidin 3-hydroxylase, from the luminescent fungus (McH3H), which catalyzes the conversion of hispidin to 3-hydroxyhispidin.

Configuration of active site segments in lytic polysaccharide monooxygenases steers oxidative xyloglucan degradation.

Background: Lytic polysaccharide monooxygenases (LPMOs) are powerful enzymes that oxidatively cleave plant cell wall polysaccharides. LPMOs classified as fungal Auxiliary Activities family 9 (AA9) have been mainly studied for their activity towards cellulose; however, various members of this AA9 family have been also shown to oxidatively cleave hemicelluloses, in particularly xyloglucan (XG). So far, it has not been studied in detail how various AA9 LPMOs act in XG degradation, and in particular, how the mode-of-action relates to the structural configuration of these LPMOs.

Evidence for ligninolytic activity of the ascomycete fungus .

Background: The ascomycete fungus has been appreciated for its targeted carbohydrate-active enzymatic arsenal. As a late colonizer of herbivorous dung, the fungus acts specifically on the more recalcitrant fraction of lignocellulose and this lignin-rich biotope might have resulted in the evolution of ligninolytic activities. However, the lignin-degrading abilities of the fungus have not been demonstrated by chemical analyses at the molecular level and are, thus far, solely based on genome and secretome predictions.

Plant Aromatic Prenyltransferases: Tools for Microbial Cell Factories.

In plants, prenylation of aromatic compounds, such as (iso)flavonoids and stilbenoids, by membrane-bound prenyltransferases (PTs), is an essential step in the biosynthesis of many bioactive compounds. Prenylated aromatic compounds have various health-beneficial properties that are interesting for industrial applications, but their exploitation is limited due to their low abundance in nature. Harnessing plant aromatic PTs for prenylation in microbial cell factories may be a sustainable and economically viable alternative.

Mass spectrometric fragmentation patterns discriminate C1- and C4-oxidised cello-oligosaccharides from their non-oxidised and reduced forms.

Lytic polysaccharide monooxygenases (LPMOs) are powerful enzymes that degrade recalcitrant polysaccharides, such as cellulose. However, the identification of LPMO-generated C1- and/or C4-oxidised oligosaccharides is far from straightforward. In particular, their fragmentation patterns have not been well established when using mass spectrometry.

Tuning of p values activates substrates in flavin-dependent aromatic hydroxylases.

Hydroxylation of substituted phenols by flavin-dependent monooxygenases is the first step of their biotransformation in various microorganisms. The reaction is thought to proceed via electrophilic aromatic substitution, catalyzed by enzymatic deprotonation of substrate, in single-component hydroxylases that use flavin as a cofactor (group A). However, two-component hydroxylases (group D), which use reduced flavin as a co-substrate, are less amenable to spectroscopic investigation.

Influence of Lytic Polysaccharide Monooxygenase Active Site Segments on Activity and Affinity.

In past years, new lytic polysaccharide monooxygenases (LPMOs) have been discovered as distinct in their substrate specificity. Their unconventional, surface-exposed catalytic sites determine their enzymatic activities, while binding sites govern substrate recognition and regioselectivity. An additional factor influencing activity is the presence or absence of a family 1 carbohydrate binding module (CBM1) connected via a linker to the C-terminus of the LPMO.