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Iluud Promotor: Prof.Dr .N.C.M .Laan E Voormalighoogleraa R Ind Ebiochemi E

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Iluud Promotor: Prof.Dr .N.C.M .Laan E Voormalighoogleraa R Ind Ebiochemi E EXPLORINGTH E LIMITSO F VANILLYL-ALCOHOL OXIDASE iLuUD Promotor: Prof.dr .N.C.M .Laan e Voormalighoogleraa r ind eBiochemi e Co-promotor: Dr. W.J.H. vanBerke l Universitair docent, DepartementAgrotechnologi ee n Voedingswetenschappen, Laboratoriumvoo r Biochemie, Wageningen Universiteit Promotiecommissie: Prof.dr .A.J.R .Heck ,Universitei t vanUtrech t Prof.dr .W.M .d eVos ,Wageninge n Universiteit Prof.dr .R . Wever,Universitei t vanAmsterda m Dr.A .Mattevi ,Universit a degli Studid iPavi a t.)o<S2o\ .Z-^fc) /J RobertH.H .va nde n Heuvel EXPLORINGTH E LIMITSO F VANILLYL-ALCOHOL OXIDASE Proefschrift ter verkrijging van degraa d vandocto r opgeza gva nd erecto r magnificus vanWageninge n Universiteit, prof.dr . ir.L .Speelman , inhe t openbaar teverdedige n opvrijda g 6apri l200 1 desnamiddag st ehal ftwe e ind eAul a Contents Chapter1 Introduction: Biocatalytic production of natural vanillin andopticall ypur e compounds Chapter2 Enzymatic synthesis ofnatura l vanillin 11 Chapter3 Regio-an dstereospecifi c conversion of4-alkylphenol s 23 byth ecovalen t flavoprotein vanillyl-alcohol oxidase Chapter4 Tuning the reactivity of enzyme-bound /j-quinone 39 methidesb ymediu m engineering Chapter5 Direction of the reactivity of vanillyl-alcohol oxidase 47 with 4-alkylphenols Chapter6 Asp17 0 is crucial for the redox properties of vanillyl- 55 alcohol oxidase Chapter 7 Inversion of stereospecificity of vanillyl-alcohol 75 oxidase Chapter8 Covalent flavinylation is essential for efficient redox 89 catalysis invanillyl-alcoho l oxidase Chapter9 Detection of intact megaDalton protein assemblies of 107 vanillyl-alcohol oxidaseb ymas s spectrometry References 115 Summary 129 Samenvatting 135 Nawoord 141 Curriculumvita e 143 Listo f publications 145 ^oVW {-2.16D Stellingen behorende bij het proefschrift Exploring the limits ofvanillyl-alcoho l oxidase 1. Er is geen fundamentele reden waarom enzymen minder actief zouden zijn in niet-waterige mediada n inwaterig emedia . Klibanov,A .M .Improvin genzyme sb yusin gthe mi norgani csolvents .Nature 409 , 241-246(2001). 2. Om een winnaar te zijn moet een atleet niet alleen pijn accepteren, maar ook lereno merme et e levene nzelf s lereno me rnaa rui t te kijken. 3. Net zoals een mens het 'recht' heeft om te leven heeft een mens ook het 'recht' omt esterven . 4. Tussen humaan en Escherichia coli methylenetetrahydrofolate reductase bestaat 30% sequentie identiteit. Daarom is voorzichtigheid geboden wanneer men de interpretatie van de kristalstructuur van het Escherichiacoli enzym wil extrapoleren naarhe thuman eenzym . Guenther, B.D. et al. The structure and properties of methylenetetrahydrofolate reductase from Escherichia coli suggest how folate ameliorates human hyperhomocysteinemia.Nat. Struct. Biol. 6,359-36 5(1999) . 5. Dewereldbevolkin g valtt everdele n invluchtelinge n entoeristen . DeNapolitaans e schilder FrancescoClemente . 6. Kleuren leiden alleen maaraf , het gaat omd e contrasten. 7. Men zou vaker bij zichzelf te rade moeten gaan in plaats van verantwoordelijkheden aft ewende n naar overheidsinstanties. 8. Gezien het feit dat een verbetering van de enantiomere ratio van 1{e.e. = 0) naar 1000 (e.e. > 99%) overeenkomt met een AAGvoo r katalyse van slechts 17kJ*mor 1 is het niet verassend dat een of enkele aminozuurmutaties in een enzymgrot e effecten kunnenhebbe n opd e stereospecificiteit. May, O., Nguyen, P.T. & Arnold, F.H. Inverting enantioselectivity by directed evolution of hydantoinase for improved production of L-methionine. Nat. Biotechnol. 18,317-32 0 (2000). van den Heuvel, R.H.H., Fraaije, M.W., Ferrer, M., Mattevi,A . & van Berkel,W.J.H . Inversion of stereospecificity of vanillyl-alcohol oxidase. Proc. Natl.Acad. Sci. U.S. A. 97, 9455-9460 (2000). 9. Sporten resulteert inhog e dosesgeestelijk e enlichamelijk e energie. Chapter1 BIOCATALYTIC PRODUCTION OF NATURAL VANILLIN AND OPTICALLY PURE COMPOUNDS Chapter1 Introduction The research described in this thesis was performed within the framework of the Innovation Oriented Research Program (IOP) Catalysis of the Dutch Ministry of Economy Affairs. Together with six other projects, we were active in the Enzymatic Oxidation cluster. The aim within this cluster was to evaluate the potential of oxidative enzymes for thebiocatalyti c production ofindustriall y relevant compounds. In recent years, the interest for enzymatic processes has increased because of the rising demand for natural and environmentally friendly ingredients and products that have improved functionalities. The key advantage of enzymes is that they catalyze reactions specifically, often regio- and/or stereospecifically, and under mild conditions,thereb y saving energy. Chemical processes are often less selective and less environmentally friendly. Another important factor that has triggered the use of enzymes for the production of valuable compounds such as pharmaceuticals, fine- chemicals, and flavors and fragrances is the enormous progress made in genetic, medium, and protein engineering. These techniques provide us with the tools to overproduce and redesign enzymes, paving the way to form large amounts of the product of interest. In this chapter, we discuss the biocatalytic methods for the production of the flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde) (1) and the production of optically pure aromatic compounds. Next, we introduce the enzyme vanillyl-alcohol oxidase as being a promising candidate to produce these compounds. Production of naturalvanilli n Vanillin iswidel y used asa flavorin g compound inth e food industry.Vanilli n has also some potential medical applications, since in recent years, itha s become apparent that vanillin is an antioxidant and may have the potential to prevent oxidative damage in mammalian cells ''2. Moreover, it has been reported that vanillin is a potential agent for thetreatmen t of sickle cell anemia3. Natural vanillin is produced from glucovanillin (2) when the beans of the orchid Vanillaplanifolia are submitted to a multi-step curing procedure 4 (Fig. 1). After this curingprocess ,vanilli n isth emos t abundant component ofth e bean at aleve l of about 2-3% by weight 5'6. Other important aromatic components in the bean are 4- hydroxybenzaldehyde, 4-hydroxybenzylalcohol, and vanillic acid (4-hydroxy-3- methoxybenzoic acid). At present, only 0.4% of the world flavor market (5*10 4 kg/year out of 12*106kg/year ) isoriginatin g from V. planifolia with synthetic vanillin providing the remainder 7. Synthetic vanillin can be obtained from a number of chemical procedures using the feedstocks coniferin (glucoside of coniferyl alcohol (4- hydroxy-3-methoxycinnamyl alcohol), eugenol (4-allyl-2-methoxyphenol), lignin, or Introduction guaiacol (2-methoxyphenol) 4, and is by far cheaper than the natural product from vanillabeans . With the increasing interest in natural products alternative processes are being developed to produce natural vanillin. This is possible since the European Union legislation allows the origin of natural products deriving from living cells or their components, including incubations in which only isolated enzymes are applied 8.Th e Vanilla plant is not ideal for biotechnological purposes, as the vanillin pathway is not very actively expressed and theplan t is slowly growing 4'9. Therefore, much effort has been put in the production of vanillin metabolites using microorganisms. Microbes can be selected for their ability to grow on a precursor of vanillin as sole energy and carbon source andth e expression levels canb eraise d bymolecula r genetics. Among the potential feedstocks suggested in literature, eugenol (3), the main component of clove oil, and ferulic acid (4-hydroxy-3-methoxycinnamic acid) (4), an abundant cell-wall material, are the most promising (for recent reviews see 4'10). Eugenol catabolism to vanillin via ferulic acid hasbee n characterized inPseudomonas strains n'12 andSerratia marcescens 13(Fig . 1).I ti sinterestin g tomentio n heretha tth e first reaction inth epathwa y from eugenolt o ferulic acid, thehydroxylatio n of eugenol to coniferyl alcohol, is also catalyzed by vanillyl-alcohol oxidase from Penicillium simplicissimum l4. Vanillic acid (5) is the major product obtained from the catabolism of ferulic acid by several microorganisms like Pseudomonas 15, Streptomyces 16, and Rhodotorula 1? species. Both carboxylic acids can be used as starting material for the production of natural vanillin. It has been reported that a Nocardia strain produces vanillin from vanillic acid 18. Substantially higher levels of vanillin have been obtained with Amycolatopsis 19 and Streptomycessetonii l6. Lesage-Meessen et al. 8'20 reported a two-step bioconversion process for the production of vanillin from ferulic acid. First, ferulic acid iscatabolize d byAspergillus nigert ovanilli c acid. Second, vanillic acid is reduced byPycnoporus cinnabarinus intovanilli n (Fig. 1) Next to microbes, isolated enzymes form an attractive alternative source for the production of natural vanillin. The pungent principal of red pepper, capsaicin (8- methyl-W-vanillyl-6-nonenamide) (6) is a precursor of vanillin and can be easily obtained at relatively low cost. In the first step of the conversion of capsaicin to vanillin, capsaicin is enzymically or chemically hydrolyzed to form vanillylamine (4- hydroxy-3-methoxybenzylamine) (7) 21. This latter compound can be easily oxidized byamin e oxidases 22an dvanillyl-alcoho l oxidase 23yieldin g vanillin (Fig. 1). Chapter 1 COOH J^* N"S» D OCH3 OCH R =C4H6 —CH=CH—CH(CH
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