Function, mechanism and structure of vanillyl-alcohol oxidase Promotor: dr.N.C.M .Laan e hoogleraar in deBiochemi e Co-promotor: dr.W.J.H . van Berkel universitair docent, departementBiomoleculair e Wetenschappen, laboratorium voor Biochemie Function, mechanism and structure of vanillyl-alcohol oxidase MarcoWilhelmu s Fraaije Proefschrift ter verkrijging van degraa d van doctor op gezag van derecto r magnificus van deLandbouwuniversitei t Wageningen, dr. C.M . Karssen, in het openbaar te verdedigen opmaanda g6 apri l 1998 desnamiddag st e half twee in deAul a M.W. Fraaije -Function , mechanism and structure of vanillyl-alcohol oxidase - 1998 Dutch:'Functie ,mechanism ee n structuur van vanillyl-alcohol oxidase' ThesisWageninge n Agricultural University -Wit h summary inDutc h Cover: vanillyl-alcohol oxidase substrates (front) aromatic growth substrates for P.simplicissimum (back) Key words: Penicillium simplicissimum Ivanillyl-alcoho l oxidase/ flavin / kinetic mechanism/ immunolocalization / phenolic compounds ISBN 90-5485-828-1 Copyright © 1998 by M.W. Fraaije All rights reserved BIBLIOTHEEK LANDBOUWUNIVERSITETT WAGENINGEN MUe>82o\,2^ Stellingen 1. De cellulaire locatie van covalente flavoproteinen in eukaryoten beperkt zich niet alleen tot specifieke organellen. Otto, A. et al., J. Biol. Chem. 271, 9823-9829, 1996. Ditproefschrift, hoofdstuk 4. 2. De vorming van een flavine-adduct tijdens flavine gekatalyseerde oxidatiereacties hoeft geen bewijs tezij n vooree ncarbanionmechanisme . Porter et al., J. Biol. Chem. 248, 4400-4416, 1973. Ditproefschrift, hoofdstuk 8. Detopologi e van hetFAD-binden d domein van vanillyl-alcohol oxidase istyperen d vooree n nieuwefamili e vanflavine-afhankelijk e oxidoreductases. Ditproefschrift, hoofdstuk 11. De afkorting VAO (vanillyl-alcohol oxidase) zou ook kunnen staan voor Various Aromats Oxidase. Dit proefschrift, hoofdstuk 5 en 7. Penicillium simplicissimum is noch een antibioticum noch een bacterie doch simpelweg een schimmel. Otto, A. et al., J. Biol. Chem. 271, 9823-9829, 1996. Johnsson, K. et al., /. Biol. Chem. 272, 2834-2840, 1997. 6. De aanname van Mayfield en Duvall dat catalase-peroxidases enkel in prokaryoten voorkomen, gaat voorbij aan bestaande literatuur en derhalve is de door hen voorgestelde evolutionaire stamboomva ndez eenzyme n incorrect. Mayfield, J.E. & Duvall, M.R., J. Mol. Evolution 42, 469-471, 1996. 7. Het ontbreken vanfossiel e paddestoelen inpalaeontologisch e collectiesi s naast een geringe belangstelling voor deze 'lagere' organismen ook te wijten aan de samenstelling vand evruchtlichamen . Fraaije, M.W. & Fraaije, R.H.B., Contr. Tert. Quatern. Geol. 32, 27-33, 1995. 8. Doorhe t toenemendemisbrui k vane-mai l adressen voorreclamedoeleinde n zou ook ope-mai l adressenee nja/nee-sticke ropti emoete nzitten . Intermediair, 11decembe r 1997 9. Collegialiteit enee noneindig e drangnaa ree n gevoel van eigenwaarde gaan moeilijk samen. 10. Zinloosgewel d isee n lozekreet . 11. Tijdelijke aanstellingen zijn slechtvoo rhe tmilieu . Stellingen behorende bij het proefschrift 'Function, mechanism and structure of vanillyl-alcohol oxidase' MarcoW . Fraaije Wageningen, 6apri l 1998 Contents Chapter 1 General introduction 1 Chapter 2 Enigmatic gratuitous induction of thecovalen t flavoprotein 15 vanillyl-alcohol oxidasei nPenicillium simplicissimum Chapter 3 Purification andcharacterizatio n of anintracellula r 29 catalase-peroxidase from Penicillium simplicissimum Chapter 4 Subcellular localization of vanillyl-alcohol oxidase 45 inPenicillium simplicissimum Chapter 5 Substrate specificity of flavin-dependent vanillyl-alcohol 57 oxidase from Penicillium simplicissimum Chapter 6 Catalytic mechanism ofth e oxidative demethylation of 73 4-(methoxymethyl)phenol by vanillyl-alcohol oxidase Chapter 7 Enantioselective hydroxylation of 4-alkylphenols 91 by vanillyl-alcohol oxidase Chapter 8 Kinetic mechanism of vanillyl-alcohol oxidase with 105 short-chain 4-alkylphenols Chapter 9 Mercuration of vanillyl-alcohol oxidasefro m 121 Penicillium simplicissimum generates inactive dimers Chapter 10 Crystal structures and inhibitor binding in the octameric 129 flavoenzyme vanillyl-alcohol oxidase Chapter 11 Anove l oxidoreductase family sharing aconserve d 153 FAD binding domain Abbreviations and nomenclature 161 Summary 163 Samenvatting 171 Curriculum vitae 177 List of publications 179 Dankwoord 181 1 General introduction 1.1. Flavoenzymes Riboflavin (vitamin B2)i s anessentia l vitamin for many life forms. Only plants and bacteria are able toproduc e this yellow (lat: flavus =yellow ) pigment denovo. Mammals , on the other hand, have to take up riboflavin in their food. In humans, riboflavin is absorbed by the gut and is transported, partially bound to serum albumin, via the bloodstream (Decker, 1994). By the subsequent action of flavokinase and FAD synthase, riboflavin isconverte d intoth ecofactors : flavin mononucleotide (FMN) andflavi n adenine dinucleotide (FAD), respectively (Scheme 1). These cofactors are incorporated into the so-called flavoenzymes thatnee dthes ecofactor s for their activity.A strikin gfeatur e of this group of enzymes isth e diversity of reactions that are catalyzed which ranges from redox catalysis andDN Arepai rt o light emission (Ghisla andMassey , 1989). Scheme 1. Structure of riboflavin (1), FMN (2) and FAD (3) in oxidized and fully reduced state. Chapter1 1.2. Flavoprotein oxidases 1.2.1. Reaction mechanism Biological oxidation reactions mostly involve the rupture of a C-H bond with a concomitant transfer of twoelectron st oa nelectro n acceptor likeNAD(P) +,cytochromes ,o r FAD. Flavin-mediated oxidation reactions vary from relatively simple oxidation reactions, like alcohol oxidations,t omor ecomple x reactions,e.g . stereoselective oxidative cyclization of plant alkaloids (Kutchan andDittrich , 1995). E-FAD S + 02 - P + H202 (1) Reactions catalyzed by flavoprotein oxidases generally include two substrates (Equation 1): an electron donor (substrate) and molecular oxygen. Therefore, these enzymatic reactions can be characterized as two-substrate two-product reactions. As a consequence, most flavoprotein oxidases obey aping-pon g mechanism ora ternar y complex mechanism. The type of kinetic mechanism varies between different flavoprotein oxidases and can depend on the type of substrate (Ramsay, 1991;Fraaij e and van Berkel, 1997). Kinetic analysis of the different enzymes hasreveale d thatth e rate-limiting stepi n catalysis is often represented by either the rate of flavin reduction or the rate of product release. As for the type of mechanism, the rate-limiting step may vary depending on the type of substrate.Furthermore , in the case ofD-amin o acid oxidase, itha sbee n found that with the yeast enzyme therat eo f flavin reduction israte-limitin g while withth e mammalian enzyme product release limits turnover (Pollegioni et al., 1993; Vanoni et al., 1997). Taken together, flavoprotein oxidases arequit e variable with respect tothei r kineticproperties . Flavoprotein oxidase catalysis involves two half-reactions in which first the flavin is reduced by the electron donor (substrate) (reductive half-reaction) and subsequently the reduced flavin is reoxidized by molecular oxygen (oxidative half-reaction). Using the stopped-flow technique,thes etw ohalf-reaction s can beanalyze d separately. - reductive half-reaction + E0x S -«— Eox ~S-«— EreCj~ P *• EreC|+ P (2) k-i k_2 By anaerobically mixing the oxidized enzyme with substrate, the reductive half- reaction can be studied. Thereactio n includes thefirs t step in thecatalyti c cycle:bindin go f substrate to form the Michaelis-Menten complex (E0x~S) (Equation 1).I n the succeeding Introduction step the flavin cofactor is reduced by the transfer of two electrons from the substrate. The mechanism by which these electrons are transferred to the N5 atom of the flavin isoalloxazine ring is still a matter of discussion. Three types of mechanisms have been proposed. A carbanion mechanism is thought to be operative when the C-H bond to be oxidized is activated. In that case, the reaction proceeds by abstraction of ahydroge n as a proton to form a carbanion. Subsequently, a N5 flavin adduct is transiently formed by which the electrons are transferred to the flavin (Porter et al., 1973).A radica l mechanism involvingth etransfe r of aproto n andtw osingl eelectron sfro m the 'non-activated' substrate toth e oxidized flavin hasbee n proposed for e.g. monoamine oxidase andmethano l oxidase (Silverman, 1995;Sherr y and Abeles, 1985). Another alternative mechanism is the direct transfer of a hydride to the flavin ring system similar to the well-established reduction of pyridine nucleotides (Mattevi et al., 1996; Pollegioni et al., 1997). Depending on the enzyme, the formed product or product intermediate may be released from the active-site before the reduced flavin is reoxidized (£3) by molecular oxygen. In case of a ternary complex mechanism, this stepwil lb eo fn o significance. - oxidative half-reaction Ered(~L) +0 2 -^ Eox (~L) +H 202 (i Eox +L ) (3) (Lrepresent s substrate, product or other bound ligand) The oxidative half-reaction can be examined by mixing the reduced enzyme with molecular oxygen. Reduced flavoprotein oxidases generally react rapidly with molecular oxygen yielding oxidized enzyme andhydroge n peroxide (£4).Contrar y to flavin-dependent monooxygenases, no oxygenated flavin intermediates have ever been detected during this relatively fast process (Massey, 1994). In flavoprotein dehydrogenases,