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1.
Biomolecules ; 11(11)2021 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-34827630

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes secreted by many organisms and viruses. LPMOs catalyze the oxidative cleavage of different types of polysaccharides and are today divided into eight families (AA9-11, AA13-17) within the Auxiliary Activity enzyme class of the CAZy database. LPMOs minimal architecture encompasses a catalytic domain, to which can be appended a carbohydrate-binding module. Intriguingly, we observed that some LPMO sequences also display a C-terminal extension of varying length not associated with any known function or fold. Here, we analyzed 27,060 sequences from different LPMO families and show that 60% have a C-terminal extension predicted to be intrinsically disordered. Our analysis shows that these disordered C-terminal regions (dCTRs) are widespread in all LPMO families (except AA13) and differ in terms of sequence length and amino-acid composition. Noteworthily, these dCTRs have so far only been observed in LPMOs. LPMO-dCTRs share a common polyampholytic nature and an enrichment in serine and threonine residues, suggesting that they undergo post-translational modifications. Interestingly, dCTRs from AA11 and AA15 are enriched in redox-sensitive, conditionally disordered regions. The widespread occurrence of dCTRs in LPMOs from evolutionarily very divergent organisms, hints at a possible functional role and opens new prospects in the field of LPMOs.

2.
Appl Environ Microbiol ; 87(24): e0152621, 2021 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-34613753

RESUMO

Copper radical alcohol oxidases (CRO-AlcOx), which have been recently discovered among fungal phytopathogens, are attractive for the production of fragrant fatty aldehydes. With the initial objective to investigate the secretion of CRO-AlcOx by natural fungal strains, we undertook time course analyses of the secretomes of three Colletotrichum species (C. graminicola, C. tabacum, and C. destructivum) using proteomics. The addition of a copper-manganese-ethanol mixture in the absence of any plant-biomass mimicking compounds to Colletotrichum cultures unexpectedly induced the secretion of up to 400 proteins, 29 to 52% of which were carbohydrate-active enzymes (CAZymes), including a wide diversity of copper-containing oxidoreductases from the auxiliary activities (AA) class (AA1, AA3, AA5, AA7, AA9, AA11, AA12, AA13, and AA16). Under these specific conditions, while a CRO-glyoxal oxidase from the AA5_1 subfamily was among the most abundantly secreted proteins, the targeted AA5_2 CRO-AlcOx were secreted at lower levels, suggesting heterologous expression as a more promising strategy for CRO-AlcOx production and utilization. C. tabacum and C. destructivum CRO-AlcOx were thus expressed in Pichia pastoris, and their preference toward both aromatic and aliphatic primary alcohols was assessed. The CRO-AlcOx from C. destructivum was further investigated in applied settings, revealing a full conversion of C6 and C8 alcohols into their corresponding fragrant aldehydes. IMPORTANCE In the context of the industrial shift toward greener processes, the biocatalytic production of aldehydes is of utmost interest owing to their importance for their use as flavor and fragrance ingredients. Copper radical alcohol oxidases (CRO-AlcOx) have the potential to become platform enzymes for the oxidation of alcohols to aldehydes. However, the secretion of CRO-AlcOx by natural fungal strains has never been explored, while the use of crude fungal secretomes is an appealing approach for industrial applications to alleviate various costs pertaining to biocatalyst production. While investigating this primary objective, the secretomics studies revealed unexpected results showing that under the oxidative stress conditions we probed, Colletotrichum species can secrete a broad diversity of copper-containing enzymes (laccases, sugar oxidoreductases, and lytic polysaccharide monooxygenases [LPMOs]) usually assigned to "plant cell wall degradation," despite the absence of any plant-biomass mimicking compound. However, in these conditions, only small amounts of CRO-AlcOx were secreted, pointing out recombinant expression as the most promising path for their biocatalytic application.

3.
Commun Biol ; 4(1): 871, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34267314

RESUMO

Fungal biotechnology is set to play a keystone role in the emerging bioeconomy, notably to address pollution issues arising from human activities. Because they preserve biological diversity, Biological Resource Centres are considered as critical infrastructures to support the development of biotechnological solutions. Here, we report the first large-scale phenotyping of more than 1,000 fungal strains with evaluation of their growth and degradation potential towards five industrial, human-designed and recalcitrant compounds, including two synthetic dyes, two lignocellulose-derived compounds and a synthetic plastic polymer. We draw a functional map over the phylogenetic diversity of Basidiomycota and Ascomycota, to guide the selection of fungal taxa to be tested for dedicated biotechnological applications. We evidence a functional diversity at all taxonomic ranks, including between strains of a same species. Beyond demonstrating the tremendous potential of filamentous fungi, our results pave the avenue for further functional exploration to solve the ever-growing issue of ecosystems pollution.


Assuntos
Biotecnologia/métodos , Corantes/metabolismo , Fungos/metabolismo , Microbiologia Industrial/métodos , Lignina/metabolismo , Plásticos/metabolismo , Ascomicetos/classificação , Ascomicetos/genética , Ascomicetos/metabolismo , Basidiomycota/classificação , Basidiomycota/genética , Basidiomycota/metabolismo , Fungos/classificação , Fungos/genética , Variação Genética , Geografia , Humanos , Fenótipo , Filogenia , Especificidade da Espécie
4.
Appl Environ Microbiol ; 87(19): e0052921, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34319813

RESUMO

The fish pathogen Aliivibrio (Vibrio) salmonicida LFI1238 is thought to be incapable of utilizing chitin as a nutrient source, since approximately half of the genes representing the chitinolytic pathway are disrupted by insertion sequences. In the present study, we combined a broad set of analytical methods to investigate this hypothesis. Cultivation studies revealed that A. salmonicida grew efficiently on N-acetylglucosamine (GlcNAc) and chitobiose [(GlcNAc)2], the primary soluble products resulting from enzymatic chitin hydrolysis. The bacterium was also able to grow on chitin particles, albeit at a lower rate than on the soluble substrates. The genome of the bacterium contains five disrupted chitinase genes (pseudogenes) and three intact genes encoding a glycoside hydrolase family 18 (GH18) chitinase and two auxiliary activity family 10 (AA10) lytic polysaccharide monooxygenases (LPMOs). Biochemical characterization showed that the chitinase and LPMOs were able to depolymerize both α- and ß-chitin to (GlcNAc)2 and oxidized chitooligosaccharides, respectively. Notably, the chitinase displayed up to 50-fold lower activity than other well-studied chitinases. Deletion of the genes encoding the intact chitinolytic enzymes showed that the chitinase was important for growth on ß-chitin, whereas the LPMO gene deletion variants only showed minor growth defects on this substrate. Finally, proteomic analysis of A. salmonicida LFI1238 growth on ß-chitin showed expression of all three chitinolytic enzymes and, intriguingly, also three of the disrupted chitinases. In conclusion, our results show that A. salmonicida LFI1238 can utilize chitin as a nutrient source and that the GH18 chitinase and the two LPMOs are needed for this ability. IMPORTANCE The ability to utilize chitin as a source of nutrients is important for the survival and spread of marine microbial pathogens in the environment. One such pathogen is Aliivibrio (Vibrio) salmonicida, the causative agent of cold water vibriosis. Due to extensive gene decay, many key enzymes in the chitinolytic pathway have been disrupted, putatively rendering this bacterium incapable of chitin degradation and utilization. In the present study, we demonstrate that A. salmonicida can degrade and metabolize chitin, the most abundant biopolymer in the ocean. Our findings shed new light on the environmental adaption of this fish pathogen.


Assuntos
Aliivibrio salmonicida/metabolismo , Quitina/metabolismo , Acetilglucosamina/metabolismo , Aliivibrio salmonicida/genética , Animais , Quitinases/genética , Quitinases/metabolismo , Dissacarídeos/metabolismo , Peixes , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Transdução de Sinais
5.
Biotechnol Adv ; : 107787, 2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34147589

RESUMO

From Egyptian mummies to the Chanel n°5 perfume, fatty aldehydes have long been used and keep impacting our senses in a wide range of foods, beverages and perfumes. Natural sources of fatty aldehydes are threatened by qualitative and quantitative variability while traditional chemical routes are insufficient to answer the society shift toward more sustainable and natural products. The production of fatty aldehydes using biotechnologies is therefore the most promising alternative for the flavors and fragrances industry. In this review, after drawing the portrait of the origin and characteristics of fragrant fatty aldehydes, we present the three main classes of enzymes that catalyze the reaction of fatty alcohols oxidation into aldehydes, namely alcohol dehydrogenases, flavin-dependent alcohol oxidases and copper radical alcohol oxidases. The constraints, challenges and opportunities to implement these oxidative enzymes in the flavors and fragrances industry are then discussed. By setting the scene on the biocatalytic production of fatty aldehydes, and providing a critical assessment of its potential, we expect this review to contribute to the development of biotechnology-based solutions in the flavors and fragrances industry.

6.
Nat Commun ; 12(1): 2132, 2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33837197

RESUMO

Oxidative plant cell-wall processing enzymes are of great importance in biology and biotechnology. Yet, our insight into the functional interplay amongst such oxidative enzymes remains limited. Here, a phylogenetic analysis of the auxiliary activity 7 family (AA7), currently harbouring oligosaccharide flavo-oxidases, reveals a striking abundance of AA7-genes in phytopathogenic fungi and Oomycetes. Expression of five fungal enzymes, including three from unexplored clades, expands the AA7-substrate range and unveils a cellooligosaccharide dehydrogenase activity, previously unknown within AA7. Sequence and structural analyses identify unique signatures distinguishing the strict dehydrogenase clade from canonical AA7 oxidases. The discovered dehydrogenase directly is able to transfer electrons to an AA9 lytic polysaccharide monooxygenase (LPMO) and fuel cellulose degradation by LPMOs without exogenous reductants. The expansion of redox-profiles and substrate range highlights the functional diversity within AA7 and sets the stage for harnessing AA7 dehydrogenases to fine-tune LPMO activity in biotechnological conversion of plant feedstocks.


Assuntos
Celulose/metabolismo , Proteínas Fúngicas/metabolismo , Oomicetos/enzimologia , Oxirredutases/metabolismo , Parede Celular/química , Parede Celular/metabolismo , Cristalografia por Raios X , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Flavoproteínas Transferidoras de Elétrons/metabolismo , Ensaios Enzimáticos , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/ultraestrutura , Microbiologia Industrial/métodos , Espectroscopia de Ressonância Magnética , Oomicetos/genética , Oxirredução , Oxirredutases/genética , Oxirredutases/isolamento & purificação , Oxirredutases/ultraestrutura , Filogenia , Análise de Sequência de DNA , Especificidade por Substrato
7.
N Biotechnol ; 62: 68-78, 2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-33524585

RESUMO

The use of retaining glycoside hydrolases as synthetic tools for glycochemistry is highly topical and the focus of considerable research. However, due to the incomplete identification of the molecular determinants of the transglycosylation/hydrolysis partition (t/h), rational engineering of retaining glycoside hydrolases to create transglycosylases remains challenging. Therefore, to understand better the factors that underpin transglycosylation in a GH51 retaining α-l-arabinofuranosidase from Thermobacillus xylanilyticus, the investigation of this enzyme's active site was pursued. Specifically, the properties of two mutants, F26L and L352M, located in the vicinity of the active site are described, using kinetic and 3D structural analyses and molecular dynamics simulations. The results reveal that the presence of L352M in the context of a triple mutant (also containing R69H and N216W) generates changes both in the donor and acceptor subsites, the latter being the result of a domino-like effect. Overall, the mutant R69H-N216W-L352M displays excellent transglycosylation activity (70 % yield, 78 % transfer rate and reduced secondary hydrolysis of the product). In the course of this study, the central role played by the conserved R69 residue was also reaffirmed. The mutation R69H affects both the catalytic nucleophile and the acid/base, including their flexibility, and has a determinant effect on the t/h partition. Finally, the results reveal that increased loop flexibility in the acceptor subsites creates new interactions with the acceptor, in particular with a hydrophobic binding platform composed of N216W, W248 and W302.


Assuntos
Glicosídeo Hidrolases/metabolismo , Cristalografia por Raios X , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosilação , Hidrólise , Modelos Moleculares , Mutação
8.
Nat Commun ; 11(1): 5786, 2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-33188177

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) are widely distributed in Nature, where they catalyze the hydroxylation of glycosidic bonds in polysaccharides. Despite the importance of LPMOs in the global carbon cycle and in industrial biomass conversion, the catalytic properties of these monocopper enzymes remain enigmatic. Strikingly, there is a remarkable lack of kinetic data, likely due to a multitude of experimental challenges related to the insoluble nature of LPMO substrates, like cellulose and chitin, and to the occurrence of multiple side reactions. Here, we employed competition between well characterized reference enzymes and LPMOs for the H2O2 co-substrate to kinetically characterize LPMO-catalyzed cellulose oxidation. LPMOs of both bacterial and fungal origin showed high peroxygenase efficiencies, with kcat/KmH2O2 values in the order of 105-106 M-1 s-1. Besides providing crucial insight into the cellulolytic peroxygenase reaction, these results show that LPMOs belonging to multiple families and active on multiple substrates are true peroxygenases.


Assuntos
Celulose/metabolismo , Oxigenases de Função Mista/metabolismo , Bactérias/enzimologia , Catalase/metabolismo , Quitina/metabolismo , Fungos/enzimologia , Peroxidase do Rábano Silvestre/metabolismo , Peróxido de Hidrogênio/metabolismo , Cinética , Nanopartículas/química , Especificidade por Substrato
9.
Nat Commun ; 11(1): 890, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-32060276

RESUMO

Lytic polysaccharide (mono)oxygenases (LPMOs) perform oxidative cleavage of polysaccharides, and are key enzymes in biomass processing and the global carbon cycle. It has been shown that LPMO reactions may be driven by light, using photosynthetic pigments or photocatalysts, but the mechanism behind this highly attractive catalytic route remains unknown. Here, prompted by the discovery that LPMOs catalyze a peroxygenase reaction more efficiently than a monooxygenase reaction, we revisit these light-driven systems, using an LPMO from Streptomyces coelicolor (ScAA10C) as model cellulolytic enzyme. By using coupled enzymatic assays, we show that H2O2 is produced and necessary for efficient light-driven activity of ScAA10C. Importantly, this activity is achieved without addition of reducing agents and proportional to the light intensity. Overall, the results highlight the importance of controlling fluxes of reactive oxygen species in LPMO reactions and demonstrate the feasibility of light-driven, tunable enzymatic peroxygenation to degrade recalcitrant polysaccharides.


Assuntos
Celulose/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Oxigenases/química , Oxigenases/metabolismo , Streptomyces coelicolor/enzimologia , Biocatálise , Celulose/química , Estabilidade Enzimática , Proteínas Fúngicas/genética , Peróxido de Hidrogênio/metabolismo , Cinética , Luz , Oxigenases/genética , Polimerização/efeitos da radiação , Streptomyces coelicolor/química , Streptomyces coelicolor/genética , Streptomyces coelicolor/efeitos da radiação
10.
Proc Natl Acad Sci U S A ; 117(3): 1504-1513, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31907317

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of monocopper enzymes broadly distributed across the tree of life. Recent reports indicate that LPMOs can use H2O2 as an oxidant and thus carry out a novel type of peroxygenase reaction involving unprecedented copper chemistry. Here, we present a combined computational and experimental analysis of the H2O2-mediated reaction mechanism. In silico studies, based on a model of the enzyme in complex with a crystalline substrate, suggest that a network of hydrogen bonds, involving both the enzyme and the substrate, brings H2O2 into a strained reactive conformation and guides a derived hydroxyl radical toward formation of a copper-oxyl intermediate. The initial cleavage of H2O2 and subsequent hydrogen atom abstraction from chitin by the copper-oxyl intermediate are the main energy barriers. Stopped-flow fluorimetry experiments demonstrated that the priming reduction of LPMO-Cu(II) to LPMO-Cu(I) is a fast process compared to the reoxidation reactions. Using conditions resulting in single oxidative events, we found that reoxidation of LPMO-Cu(I) is 2,000-fold faster with H2O2 than with O2, the latter being several orders of magnitude slower than rates reported for other monooxygenases. The presence of substrate accelerated reoxidation by H2O2, whereas reoxidation by O2 became slower, supporting the peroxygenase paradigm. These insights into the peroxygenase nature of LPMOs will aid in the development and application of enzymatic and synthetic copper catalysts and contribute to a further understanding of the roles of LPMOs in nature, varying from biomass conversion to chitinolytic pathogenesis-defense mechanisms.


Assuntos
Proteínas de Bactérias/metabolismo , Quitina/metabolismo , Oxigenases de Função Mista/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Celulose/química , Celulose/metabolismo , Quitina/química , Cobre/química , Cobre/metabolismo , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Oxirredução , Serratia marcescens/enzimologia
11.
Nat Chem Biol ; 16(3): 345-350, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31932718

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that play a key role in the oxidative degradation of various biopolymers such as cellulose and chitin. While hunting for new LPMOs, we identified a new family of proteins, defined here as X325, in various fungal lineages. The three-dimensional structure of X325 revealed an overall LPMO fold and a His brace with an additional Asp ligand to Cu(II). Although LPMO-type activity of X325 members was initially expected, we demonstrated that X325 members do not perform oxidative cleavage of polysaccharides, establishing that X325s are not LPMOs. Investigations of the biological role of X325 in the ectomycorrhizal fungus Laccaria bicolor revealed exposure of the X325 protein at the interface between fungal hyphae and tree rootlet cells. Our results provide insights into a family of copper-containing proteins, which is widespread in the fungal kingdom and is evolutionarily related to LPMOs, but has diverged to biological functions other than polysaccharide degradation.


Assuntos
Cobre/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Sítios de Ligação , Celulose/metabolismo , Quitina/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Fungos/metabolismo , Oxigenases de Função Mista/ultraestrutura , Oxirredução , Filogenia , Polissacarídeos/metabolismo
12.
FEBS J ; 287(5): 897-908, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31532909

RESUMO

The catalytic function of lytic polysaccharide monooxygenases (LPMOs) to cleave and decrystallize recalcitrant polysaccharides put these enzymes in the spotlight of fundamental and applied research. Here we demonstrate that the demand of LPMO for an electron donor and an oxygen species as cosubstrate can be fulfilled by a single auxiliary enzyme: an engineered fungal cellobiose dehydrogenase (CDH) with increased oxidase activity. The engineered CDH was about 30 times more efficient in driving the LPMO reaction due to its 27 time increased production of H2 O2 acting as a cosubstrate for LPMO. Transient kinetic measurements confirmed that intra- and intermolecular electron transfer rates of the engineered CDH were similar to the wild-type CDH, meaning that the mutations had not compromised CDH's role as an electron donor. These results support the notion of H2 O2 -driven LPMO activity and shed new light on the role of CDH in activating LPMOs. Importantly, the results also demonstrate that the use of the engineered CDH results in fast and steady LPMO reactions with CDH-generated H2 O2 as a cosubstrate, which may provide new opportunities to employ LPMOs in biomass hydrolysis to generate fuels and chemicals.


Assuntos
Desidrogenases de Carboidrato/metabolismo , Oxigenases de Função Mista/metabolismo , Polissacarídeos/metabolismo , Celulose/metabolismo , Peróxido de Hidrogênio/metabolismo
13.
J Biol Chem ; 294(50): 19349-19364, 2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31656228

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) catalyze oxidative cleavage of recalcitrant polysaccharides such as cellulose and chitin and play an important role in the enzymatic degradation of biomass. Although it is clear that these monocopper enzymes have extended substrate-binding surfaces for interacting with their fibrous substrates, the structural determinants of LPMO substrate specificity remain largely unknown. To gain additional insight into substrate specificity in LPMOs, here we generated a mutant library of a cellulose-active family AA10 LPMO from Streptomyces coelicolor A3(2) (ScLPMO10C, also known as CelS2) having multiple substitutions at five positions on the substrate-binding surface that we identified by sequence comparisons. Screening of this library using a newly-developed MS-based high-throughput assay helped identify multiple enzyme variants that contained four substitutions and exhibited significant chitinolytic activity and a concomitant decrease in cellulolytic activity. The chitin-active variants became more rapidly inactivated during catalysis than a natural chitin-active AA10 LPMO, an observation likely indicative of suboptimal substrate binding leading to autocatalytic oxidative damage of these variants. These results reveal several structural determinants of LPMO substrate specificity and underpin the notion that productive substrate binding by these enzymes is complex, depending on a multitude of amino acids located on the substrate-binding surface.


Assuntos
Celulose/metabolismo , Quitina/metabolismo , Oxigenases de Função Mista/metabolismo , Polissacarídeos/metabolismo , Engenharia de Proteínas , Streptomyces coelicolor/enzimologia , Oxigenases de Função Mista/genética , Modelos Moleculares , Especificidade por Substrato
14.
Curr Opin Struct Biol ; 59: 54-64, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30947104

RESUMO

The discovery of oxidative cleavage of glycosidic bonds by enzymes currently known as lytic polysaccharide monooxygenases (LPMOs) has had a major impact on our current understanding of the enzymatic conversion of recalcitrant polysaccharides such as chitin and cellulose. The number of LPMO sequence families keeps expanding and novel substrate specificities and biological functionalities are being discovered. The catalytic mechanism of these LPMOs remains somewhat enigmatic. Recently, novel insights have been obtained from studies of enzyme-substrate complexes by X-ray crystallography, EPR, NMR, and modeling. Furthermore, it has been shown that LPMOs may carry out peroxygenase reactions, at much higher rates than monooxygenase reactions, which affects our understanding and exploitation of these powerful enzymes.


Assuntos
Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Catálise , Domínio Catalítico , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Hidrólise , Oxigenases de Função Mista/genética , Oxirredução , Oxigênio/metabolismo , Filogenia , Ligação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato
15.
Biotechnol Biofuels ; 12: 58, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30923566

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) are abundant in nature and best known for their role in the enzymatic conversion of recalcitrant polysaccharides such as chitin and cellulose. LPMO activity requires an oxygen co-substrate, which was originally thought to be O2, but which may also be H2O2. Functional characterization of LPMOs is not straightforward because typical reaction mixtures will promote side reactions, including auto-catalytic inactivation of the enzyme. For example, despite some recent progress, there is still limited insight into the kinetics of the LPMO reaction. Recent discoveries concerning the role of H2O2 in LPMO catalysis further complicate the picture. Here, we review commonly used methods for characterizing LPMOs, with focus on benefits and potential pitfalls, rather than on technical details. We conclude by pointing at a few key problems and potential misconceptions that should be taken into account when interpreting existing data and planning future experiments.

16.
Appl Environ Microbiol ; 85(5)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30578267

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that perform oxidative cleavage of recalcitrant polysaccharides. We have purified and characterized a recombinant family AA9 LPMO, LPMO9B, from Gloeophyllum trabeum (GtLPMO9B) which is active on both cellulose and xyloglucan. Activity of the enzyme was tested in the presence of three different reductants: ascorbic acid, gallic acid, and 2,3-dihydroxybenzoic acid (2,3-DHBA). Under standard aerobic conditions typically used in LPMO experiments, the first two reductants could drive LPMO catalysis whereas 2,3-DHBA could not. In agreement with the recent discovery that H2O2 can drive LPMO catalysis, we show that gradual addition of H2O2 allowed LPMO activity at very low, substoichiometric (relative to products formed) reductant concentrations. Most importantly, we found that while 2,3-DHBA is not capable of driving the LPMO reaction under standard aerobic conditions, it can do so in the presence of externally added H2O2 At alkaline pH, 2,3-DHBA is able to drive the LPMO reaction without externally added H2O2, and this ability overlaps entirely the endogenous generation of H2O2 by GtLPMO9B-catalyzed oxidation of 2,3-DHBA. These findings support the notion that H2O2 is a cosubstrate of LPMOs and provide insight into how LPMO reactions depend on, and may be controlled by, the choice of pH and reductant.IMPORTANCE Lytic polysaccharide monooxygenases promote enzymatic depolymerization of lignocellulosic materials by microorganisms due to their ability to oxidatively cleave recalcitrant polysaccharides. The properties of these copper-dependent enzymes are currently of high scientific and industrial interest. We describe a previously uncharacterized fungal LPMO and show how reductants, which are needed to prime the LPMO by reducing Cu(II) to Cu(I) and to supply electrons during catalysis, affect enzyme efficiency and stability. The results support claims that H2O2 is a natural cosubstrate for LPMOs by demonstrating that when certain reductants are used, catalysis can be driven only by H2O2 and not by O2 Furthermore, we show how auto-inactivation resulting from endogenous generation of H2O2 in the LPMO-reductant system may be prevented. Finally, we identified a reductant that leads to enzyme activation without any endogenous H2O2 generation, allowing for improved control of LPMO reactivity and providing a valuable tool for future LPMO research.


Assuntos
Basidiomycota/metabolismo , Peróxido de Hidrogênio/metabolismo , Oxigenases de Função Mista/metabolismo , Polissacarídeos/metabolismo , Basidiomycota/genética , Celulase/metabolismo , Celulose/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glucanos/metabolismo , Concentração de Íons de Hidrogênio , Lignina/metabolismo , Oxigenases de Função Mista/genética , Oxirredução , Pichia/genética , Substâncias Redutoras , Madeira , Xilanos/metabolismo
17.
J Biol Chem ; 294(5): 1516-1528, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30514757

RESUMO

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that catalyze oxidative cleavage of glycosidic bonds in polysaccharides in the presence of an external electron donor (reductant). In the classical O2-driven monooxygenase reaction, the reductant is needed in stoichiometric amounts. In a recently discovered, more efficient H2O2-driven reaction, the reductant would be needed only for the initial reduction (priming) of the LPMO to its catalytically active Cu(I) form. However, the influence of the reductant on reducing the LPMO or on H2O2 production in the reaction remains undefined. Here, we conducted a detailed kinetic characterization to investigate how the reductant affects H2O2-driven degradation of 14C-labeled chitin by a bacterial LPMO, SmLPMO10A (formerly CBP21). Sensitive detection of 14C-labeled products and careful experimental set-ups enabled discrimination between the effects of the reductant on LPMO priming and other effects, in particular enzyme-independent production of H2O2 through reactions with O2 When supplied with H2O2, SmLPMO10A catalyzed 18 oxidative cleavages per molecule of ascorbic acid, suggesting a "priming reduction" reaction. The dependence of initial rates of chitin degradation on reductant concentration followed hyperbolic saturation kinetics, and differences between the reductants were manifested in large variations in their half-saturating concentrations (K mR app). Theoretical analyses revealed that K mR app decreases with a decreasing rate of polysaccharide-independent LPMO reoxidation (by either O2 or H2O2). We conclude that the efficiency of LPMO priming depends on the relative contributions of reductant reactivity, on the LPMO's polysaccharide monooxygenase/peroxygenase and reductant oxidase/peroxidase activities, and on reaction conditions, such as O2, H2O2, and polysaccharide concentrations.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Quitina/metabolismo , Peróxido de Hidrogênio/farmacologia , Oxigenases de Função Mista/metabolismo , Polissacarídeos Bacterianos/metabolismo , Substâncias Redutoras/farmacologia , Cinética , Oxidantes/farmacologia , Oxirredução , Especificidade por Substrato
18.
Microbiol Mol Biol Rev ; 82(4)2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30257993

RESUMO

Biomass constitutes an appealing alternative to fossil resources for the production of materials and energy. The abundance and attractiveness of vegetal biomass come along with challenges pertaining to the intricacy of its structure, evolved during billions of years to face and resist abiotic and biotic attacks. To achieve the daunting goal of plant cell wall decomposition, microorganisms have developed many (enzymatic) strategies, from which we seek inspiration to develop biotechnological processes. A major breakthrough in the field has been the discovery of enzymes today known as lytic polysaccharide monooxygenases (LPMOs), which, by catalyzing the oxidative cleavage of recalcitrant polysaccharides, allow canonical hydrolytic enzymes to depolymerize the biomass more efficiently. Very recently, it has been shown that LPMOs are not classical monooxygenases in that they can also use hydrogen peroxide (H2O2) as an oxidant. This discovery calls for a revision of our understanding of how lignocellulolytic enzymes are connected since H2O2 is produced and used by several of them. The first part of this review is dedicated to the LPMO paradigm, describing knowns, unknowns, and uncertainties. We then present different lignocellulolytic redox systems, enzymatic or not, that depend on fluxes of reactive oxygen species (ROS). Based on an assessment of these putatively interconnected systems, we suggest that fine-tuning of H2O2 levels and proximity between sites of H2O2 production and consumption are important for fungal biomass conversion. In the last part of this review, we discuss how our evolving understanding of redox processes involved in biomass depolymerization may translate into industrial applications.


Assuntos
Biotecnologia , Fungos/enzimologia , Lignina/metabolismo , Oxirredutases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Peróxido de Hidrogênio/metabolismo , Hidrólise , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Oxirredução , Plantas/química , Polissacarídeos/metabolismo
19.
Biotechnol Biofuels ; 11: 209, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30061931

RESUMO

Background: The discovery of enzymes named lytic polysaccharide monooxygenases (LPMOs) has had a major impact on the efficiency of current commercial cellulase cocktails for saccharification of lignocellulosic biomass. However, the notion that LPMOs use molecular oxygen as a co-substrate and require two externally delivered electrons per catalytic cycle poses a challenge in the development of efficient large-scale industrial processes. Building on the recent discovery that H2O2, rather than O2, is the co-substrate of LPMOs, we show here how cellulose degradation by the LPMO-containing commercial cellulase cocktail Cellic® CTec2 can be controlled and boosted by supplying the reaction with H2O2. Results: The controlled supply of anaerobic hydrolysis reactions with H2O2 and sub-stoichiometric amounts of reductant increased apparent LPMO activity by almost two orders of magnitude compared to standard aerobic reactions utilizing O2 and stoichiometric amounts of reductant. Improved LPMO activity was correlated with enhanced saccharification rates and yields for a model cellulosic substrate (Avicel) as well as industrial lignocellulosic substrates (sulfite-pulped Norway spruce and steam-exploded birch), although the magnitude of the effects was substrate dependent. Improvements in lignocellulose conversions were achieved at low H2O2 feeding rates (in the range of 90-600 µM h-1). Tight control of LPMO reactions by controlled supply of H2O2 under anaerobic conditions was possible. Conclusion: We report saccharification rates and yields for a model substrate (Avicel) and industrial lignocellulosic substrates that, at low H2O2 feeding rates, are higher than those seen under standard aerobic conditions. In an industrial setting, controlling and supplying molecular oxygen and stoichiometric amounts of reductant are challenging. The present report shows that the use of small amounts of a liquid bulk chemical, H2O2, provides an alternative to the currently available processes, which likely is cheaper and more easy to control, while giving higher product yields.

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