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A Kaleidoscopic Look at Vanillyl Alcohol Oxidase /

Record Type:	Electronic resources : Monograph/item
Title/Author:	A Kaleidoscopic Look at Vanillyl Alcohol Oxidase // Gudrun Gygli.
Author:	Gygli, Gudrun,
Description:	1 electronic resource (236 pages)
Notes:	Source: Dissertations Abstracts International, Volume: 83-04, Section: B.
Contained By:	Dissertations Abstracts International83-04B.
Subject:	Adenosine. -
Online resource:	https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=28232789
ISBN:	9798708787170

A Kaleidoscopic Look at Vanillyl Alcohol Oxidase /
Gygli, Gudrun,

A Kaleidoscopic Look at Vanillyl Alcohol Oxidase /Gudrun Gygli. - 1 electronic resource (236 pages)

Source: Dissertations Abstracts International, Volume: 83-04, Section: B.

Flavoenzymes are highly diverse enzymes that harness the power of their flavin cofactor to catalyse redox reactions. The fungal, FAD-dependent enzyme vanillyl alcohol oxidase (VAO) has been studied in this thesis through different computational and experimental approaches. This oxidative enzyme, which covalently binds its cofactor via a histidine residue, is active with a wide range of parasubstituted phenols. Several of these substrates are converted into high-value chemicals, e.g. coniferyl alcohol, vanillin and chiral alcohols.Interest in oxidative enzymes is booming and several are being prepared for industrial applications or are already used at large scale. Examples are glucose oxidase, used as a biosensor in blood glucose meters for diabetic patients; hexose oxidase, used in the baking industries to improve dough properties, and laccases, which are on their way for applications in the pulp and paper industry. Each application of a new enzyme faces different challenges. Baeyer-Villiger monooxygenases for example are promising biocatalysts for the pharmaceutical industry, but many of them are operationally unstable or suffer from substrate or product inhibition. Research to overcome these obstacles is ongoing and requires the integration of protein engineering with process design. Such integrative approaches profit from the advances made in both fields and are likely to reduce the time needed by individual enzymes to grow into their new role as industrial biocatalysts.VAO belongs to the VAO/PCMH family, whose members all share the same FAD-binding fold. Within that family, VAO is part of the subgroup of 4-phenol oxidizing enzymes. VAO is the only fungal member of that group and its physiological role in fungi is unclear. Using the sequence and three-dimensional structure of VAO from Penicillium simplicissimum, we have identified novel, putative fungal VAOs. We found that these fungal VAOs likelyoriginated from a bacterial ancestor and were transferred to fungi by horizontal gene transfer. Based on the sequences of these novel fungal VAOs we have defined several motifs that should help to resolve misannotations in sequence databases.In the reaction cycle of VAO, reoxidation of reduced FAD by dioxygen is a crucial step. After product formation, flavin reoxidation results in the release of hydrogen peroxide. VAOs that were heterologously produced in the methylotrophic yeast Komagataella phaffii contain a highly stable, non-covalently bound anionic FAD semiquinone. In VAO, this redox state of FAD is atypical and its stability is baffling. We think that enzymes produced in this eukaryotic expression system fell victim to oxidative stress: substrate binding uncouples dioxygen reduction from product formation, and thus results in atypical single electron flavin redox cycling. In other words, in these enzymes the normally quick decay of the p-quinone methide intermediate is impaired by an increased energy barrier to the product, resulting in the reaction falling back to the p-quinone methide intermediate.In any enzymatic reaction, substrates have to "find" the active site before catalysis can happen. Because most enzymes are not diffusion limited, this step is often neglected. It is nevertheless of fundamental interest to understand the inner workings of enzymes. The identification of diffusion (or migration) paths based on crystal structures remains challenging due to the rigidity of these tightly packed structures. In VAO, we have identified one path for phenolic ligands and one for dioxygen and hydrogen peroxide to enter and leave the active site. After the substrate has reached the active site, two concierge residues close off the reaction center. Interestingly, in our simulations only the protonated forms of phenolic substrates are able to enter the enzyme.A VAO-catalysed reaction that is of relevance for the food industry is the synthesis of natural vanillin from natural p-creosol. VAO catalyses this reaction via the intermediate formation of vanillyl alcohol. However, in the first reaction step, a covalent p-creosol-FAD adduct is formed, significantly inhibiting this reaction. We have used quantumchemical modelling to study this process inmore detail. The productive reaction to vanillyl alcohol competes with adduct formation due to the lower energy barrier of the reaction pathway to the adduct. An aspartate residue in the active site is crucial in adduct formation, and its flexibility is the cause for adduct formationwith p-creosol. Bulkier substrates prevent the attack of this aspartate on FAD by steric hindrance.Oxidative enzymes, such as VAO, are moving into the spotlight of different industries. One strategy to identify enzymes for industrial applications is to mine genomes. Genome mining of VAO-like sequences is hampered by misannotations present in sequence databases. Opening up sequence databases to editing by users would help to resolve misannotations.Recent experimental and computational research has further advanced the understanding of VAO's reactivity. We have demonstrated that computational tools can successfully be used to study processes out of the reach of experimental techniques. It is crucial to integrate different computationalmethodologies into one, easy to use, computational enzymology toolbox. Such a toolbox should lower the barrier between the fields of computational and experimental enzymology. Collaborations that successfully connect industry and academia, as well as computational and experimental enzymologists, will decide the future of industrial applications of enzymes.

English

ISBN: 9798708787170Subjects--Topical Terms:

1362513
Adenosine.

A Kaleidoscopic Look at Vanillyl Alcohol Oxidase /
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500 $a Source: Dissertations Abstracts International, Volume: 83-04, Section: B.
500 $a Advisors: van Berkel, W. J. H. Committee members: Stams, A. J. M.; Faulds, C. ; Fraaije, M. W.; Seebeck, F.
502 $b Ph.D. $c Wageningen University and Research $d 2018.
520 $a Flavoenzymes are highly diverse enzymes that harness the power of their flavin cofactor to catalyse redox reactions. The fungal, FAD-dependent enzyme vanillyl alcohol oxidase (VAO) has been studied in this thesis through different computational and experimental approaches. This oxidative enzyme, which covalently binds its cofactor via a histidine residue, is active with a wide range of parasubstituted phenols. Several of these substrates are converted into high-value chemicals, e.g. coniferyl alcohol, vanillin and chiral alcohols.Interest in oxidative enzymes is booming and several are being prepared for industrial applications or are already used at large scale. Examples are glucose oxidase, used as a biosensor in blood glucose meters for diabetic patients; hexose oxidase, used in the baking industries to improve dough properties, and laccases, which are on their way for applications in the pulp and paper industry. Each application of a new enzyme faces different challenges. Baeyer-Villiger monooxygenases for example are promising biocatalysts for the pharmaceutical industry, but many of them are operationally unstable or suffer from substrate or product inhibition. Research to overcome these obstacles is ongoing and requires the integration of protein engineering with process design. Such integrative approaches profit from the advances made in both fields and are likely to reduce the time needed by individual enzymes to grow into their new role as industrial biocatalysts.VAO belongs to the VAO/PCMH family, whose members all share the same FAD-binding fold. Within that family, VAO is part of the subgroup of 4-phenol oxidizing enzymes. VAO is the only fungal member of that group and its physiological role in fungi is unclear. Using the sequence and three-dimensional structure of VAO from Penicillium simplicissimum, we have identified novel, putative fungal VAOs. We found that these fungal VAOs likelyoriginated from a bacterial ancestor and were transferred to fungi by horizontal gene transfer. Based on the sequences of these novel fungal VAOs we have defined several motifs that should help to resolve misannotations in sequence databases.In the reaction cycle of VAO, reoxidation of reduced FAD by dioxygen is a crucial step. After product formation, flavin reoxidation results in the release of hydrogen peroxide. VAOs that were heterologously produced in the methylotrophic yeast Komagataella phaffii contain a highly stable, non-covalently bound anionic FAD semiquinone. In VAO, this redox state of FAD is atypical and its stability is baffling. We think that enzymes produced in this eukaryotic expression system fell victim to oxidative stress: substrate binding uncouples dioxygen reduction from product formation, and thus results in atypical single electron flavin redox cycling. In other words, in these enzymes the normally quick decay of the p-quinone methide intermediate is impaired by an increased energy barrier to the product, resulting in the reaction falling back to the p-quinone methide intermediate.In any enzymatic reaction, substrates have to "find" the active site before catalysis can happen. Because most enzymes are not diffusion limited, this step is often neglected. It is nevertheless of fundamental interest to understand the inner workings of enzymes. The identification of diffusion (or migration) paths based on crystal structures remains challenging due to the rigidity of these tightly packed structures. In VAO, we have identified one path for phenolic ligands and one for dioxygen and hydrogen peroxide to enter and leave the active site. After the substrate has reached the active site, two concierge residues close off the reaction center. Interestingly, in our simulations only the protonated forms of phenolic substrates are able to enter the enzyme.A VAO-catalysed reaction that is of relevance for the food industry is the synthesis of natural vanillin from natural p-creosol. VAO catalyses this reaction via the intermediate formation of vanillyl alcohol. However, in the first reaction step, a covalent p-creosol-FAD adduct is formed, significantly inhibiting this reaction. We have used quantumchemical modelling to study this process inmore detail. The productive reaction to vanillyl alcohol competes with adduct formation due to the lower energy barrier of the reaction pathway to the adduct. An aspartate residue in the active site is crucial in adduct formation, and its flexibility is the cause for adduct formationwith p-creosol. Bulkier substrates prevent the attack of this aspartate on FAD by steric hindrance.Oxidative enzymes, such as VAO, are moving into the spotlight of different industries. One strategy to identify enzymes for industrial applications is to mine genomes. Genome mining of VAO-like sequences is hampered by misannotations present in sequence databases. Opening up sequence databases to editing by users would help to resolve misannotations.Recent experimental and computational research has further advanced the understanding of VAO's reactivity. We have demonstrated that computational tools can successfully be used to study processes out of the reach of experimental techniques. It is crucial to integrate different computationalmethodologies into one, easy to use, computational enzymology toolbox. Such a toolbox should lower the barrier between the fields of computational and experimental enzymology. Collaborations that successfully connect industry and academia, as well as computational and experimental enzymologists, will decide the future of industrial applications of enzymes.
520 $a Enzymen maken het leven mogelijk - maar hoe doen ze dit? Enzymen versnellen (katalyseren) een chemische reactie door een omgeving te vormen waarin deze reactie sneller kan verlopen. Dit betekent ook dat de energiebarriere, die moet worden overwonnen voor elke chemische reactie, wordt verlaagd. De omgeving in het enzym waarin de reactie kan plaatsvinden wordt het actieve centrum genoemd. De interacties in het actieve centrum zijn vaak geoptimaliseerd voor een specifiek enzym-substraat molecuul paar (door natuurlijke selectie tijdens de evolutie), wat enzymen geeft met verschillende specialisaties (substraatspecificiteiten). De bouwstenen waarmee enzymen worden gebouwd (aminozuren) bepalen welk type reactie een enzym kan katalyseren.Sommige enzymen gebruiken cofactoren om reacties te katalyseren die met enkel aminozuren niet mogelijk zijn. Vitaminen zijn meestal de voorlopers voor deze cofactoren. Flavoenzymen, bijvoorbeeld het enzym vanillyl alcohol oxidase (VAO) dat is onderzocht in dit proefschrift, gebruikt een flavine cofactor om er redoxreacties mee te katalyseren. VAO is oorspronkelijk uit een schimmel geeisoleerd en bindt zijn flavine-adenine-dinucleotide covalent via een histidine-aminozuur in het actieve centrum. VAO converteert uitsluitend para-gesubstitueerde, fenolische substraten. Veel van deze substraten worden in waardevolle chemicalien zoals coniferylalcohol, vanilline en chirale alcoholen omgezet. We hebben dit enzym experimenteel in het lab en met de computer bestudeerd.De belangstelling voor oxidatieve enzymen zoals VAO is in de afgelopen jaren enorm toegenomen en veel van deze enzymen worden gebruikt of voorbereid voor industriele toepassingen. Enkele voorbeelden zijn glucoseoxidase, hexose-oxidase, laccasen of Baeyer-Villigermonooxygenasen. Glucoseoxidase is een enzym dat de bloedsuikerspiegel voor diabetici kan meten, hexose oxidase wordt in de bakkersindustrie gebruikt om de eigenschappen van het deeg te verbeteren en laccasen worden ontwikkeld voor toepassingen in de pulp- en papierindustrie. Elk enzymheeft te maken met verschillende uitdagingen en deze uitdagingen zijn afhankelijk van de gewenste toepassing. VAOs die werden geproduceerd in de gist Komagataella phaffii bevatten allemaal een hoogst ongebruikelijke en stabiele vorm van de flavine cofactor: een niet-covalent gebonden, anionische semichinon. We vermoeden dat deze enzymen het slachtoffer zijn geworden van oxidatieve stress. Door het substraat te binden, worden de normaal gekoppelde reacties die zuurstof verminderen en product vormen ontkoppeld, en de reacie eindigt zonder product te vormen. In een enzymatische reactie moeten de substraten altijd het actieve centrum van het enzym "vinden" voordat een reactie kan plaatsvinden. De migratiepaden in enzymen kunnen worden geidentificeerd door berekeningen die de bewegingen van een enzym simuleren. In VAO ontdektenwe de migratiepaden van verschillende moleculen. Twee concierge aminozuren sluiten het pad naar de actieve centrum af zodra een substraat het heeft bereikt. Interessant is dat alleen geprotoneerde substraten in in het actieve centrum worden toegelaten.De synthese van natuurlijke vanilline uit natuurlijke creosoot, gekatalyseerd door VAO, is belangrijk voor de voedingsindustrie. VAO katalyseert deze reactie met het tussenproduct vanillylalcohol. In de eerste reactiestap wordt helaas een stabiel, covalent adduct gevormd tussen het substraat en de cofactor, wat deze reactie sterk vertraagt. We hebben deze reactie in detail bestudeerd met kwantummechanische berekeningen en vonden dat de productieve reactie (waarin vanillyl alcohol wordt gevormd) concurreert met adductvorming.Oxidatieve enzymen, waaronder VAO, komen steeds meer in de schijnwerpers van verschillende industrieen. We hebben aangetoond dat computergestuurd onderzoek met succes kan worden gebruikt om processen te onderzoeken die buiten het bereik van experimentele technieken liggen. Met het oog op de toekomst van de industriele toepassingen van enzymen moet samenwerking tussen de industrie en de academischewereld worden versterkt en ook de samenwerking tussen de experimentele en computationele enzymologie moet worden uitgebreid.
546 $a English
590 $a School code: 2157
650 4 $a Adenosine. $3 1362513
650 4 $a Fungi. $3 571472
650 4 $a Oxygen. $3 714746
650 4 $a Lignin. $3 1097216
650 4 $a Oxidation. $3 714629
650 4 $a Chemical elements. $3 544626
650 4 $a Biocatalysts. $3 3683849
650 4 $a Algae. $3 546231
650 4 $a Carbon. $3 604057
650 4 $a Vitamin B. $3 3561720
650 4 $a Photosynthesis. $3 518350
650 4 $a Amino acids. $3 558768
650 4 $a Alcohol. $3 872287
650 4 $a Phenols. $3 915439
650 4 $a Cyanobacteria. $3 860872
650 4 $a Phosphorylation. $3 816803
650 4 $a Catalysis. $3 560465
650 4 $a Hydrogen peroxide. $3 1534649
650 4 $a Biochemistry. $3 518028
690 $a 0487
710 2 $a Wageningen University and Research. $e degree granting institution. $3 3815267
720 1 $a van Berkel, W. J. H. $e degree supervisor.
773 0 $t Dissertations Abstracts International $g 83-04B.
790 $a 2157
791 $a Ph.D.
792 $a 2018
856 4 0 $u https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=28232789