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Structure-Function Relationship of Flavoproteins with Special Reference to P-Hydroxybenzoate Hydroxylase from Pseudomonas Fluorescens

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Structure-Function Relationship of Flavoproteins with Special Reference to P-Hydroxybenzoate Hydroxylase from Pseudomonas Fluorescens Structure-function relationship of flavoproteins With special reference to p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens 0000 0330 0957 Promotor: dr. F. Müller, oud-hoogleraar in de Biochemie *j o^2ül, \Qrh> JU W.J . H. van Berkel Structure-function relationship of flavoproteins With special reference to p-hydroxybenzoate hydroxylase from Pseudomonasfluorescens Proefschrift ter verkrijging van de graad van doctor in de landbouwwetenschappen, op gezag van de rector magnificus, dr. H. C. van der Plas, in het openbaar te verdedigen op dinsdag 9 mei 1989 des namiddags te vier uur in de aula van de Landbouwuniversiteit te Wageningen BIBLIOTHEEK LANDBOUWUiNIVERSITEI') WAGEMNGFÏV (ôVo2?*>\} IZfo STELLINGEN 1. De enzym-nomenclatuur van FAD-bevattende monooxygenases dient nodigherzie n enu p todat egebrach t teworden . - Enzyme Nomenclature (1984)IUB ,Academi c Press (USA). - Ditproefschrift . 2. Rajue t al.gebruike n tenonrecht e deresultaten ,verkrege n inhoofdstu k 5va ndi t proefschrift,al sargumen t omaa nt e gevenda t cysteine residuen eenessentiël ero l spelen ind e katalyse van 4-hydroxyphenylacetaat-3-hydroxylase uit Pseudomonas putida. - Raju,S.G. , Kamath,A.V . and Vaidyanathan,CS . (1988) Bioch.Biophys .Res .Commun . 154,537-543 . 3. Het is jammer te moeten constateren dat "de fabrikantva n de beste biochemicaliën" anno 1989 de ratio lipoamide: diaphorase activiteit van lipoamide dehydrogenase slechts omschrijft alsee nmaa tvoo rd edenaturati e vanhe tenzym . -Massey ,V . and Veeger,C . (1961)Biochim . Biophys.Act a 48, 33-47. - Sigma catalogus (1989). 4. Zolang eenreactie-intermediai r in de natuurlijke reactie gekatalyseerd door p-hydroxybenzoaat hydroxylase nietword t gekarakteriseerd, laat staan waargenomen, blijft zijn voorkomen speculatief. - AndersonR.F. ,Patel ,K.B .an dStratford ,M.R.L . (1987) J. Biol.Chem . 262,17475-17479 . 5. Meer biochemische kennis omtrent de dehalogenering van halogeenhoudende aromatische verbindingen is dringend gewenst. - Castro,CE. ,Yokoyama ,W.H .an dBelser ,N.O . (1988) J. Agric. Food Chem. 36,915-919 . - Vande nTweel ,W.J.J . (1988)Thesis ,Wageningen . Waterstofbruggen tussen enzym ensubstraat/produkt ,zoal s bepaald via de kristalstructuur van p-hydroxybenzoaat hydroxylase,moete nme t een"korreltje "zou tgenome nworden . - Schreuder,H.A . (1988)Thesis ,Universit y ofGroningen . Helaas werkt het "verkopen" vanDNA-sequenties ,coderen d voorenzymen ,oo khe tverkope nva nonzi n ind ehand . -Matsubara ,Y. ,Kraus ,J.P. , Ozasa,H. ,Glassberg , R., Finocchiaro,G. , Ikeda,Y. ,Mole ,J. , Rosenberg,L.E .an d Tanaka,K . (1987)J . Biol. Chem. 262,10104-10108 . Het ind evoedingswerel d almee r dan tien jaar consequent vermeldenva nd e eenheidkilocalori e naastkiloJoul ewek td e schijnda td eeerstgenoemd e eenheidbete rverteerbaa ris . - Duinker-Joustra,N . (1983)Kij ko p joulese ncalorieën . De ivoren toren der wetenschap begint aardig op een vuurtoren te lijken:d eoverhei d steekt erd ebran d in! 10. Met deze stellingword t hetmees td edraa kgestoken : Stellingenbehoren d bijhe t proefschrift van Willemva nBerkel ,Wageningen , 9me i 1989. VOORWOORD Dit proefschrift ishe t resultaat van een jarenlang leerproces temidden van vele collega's en studenten. Iedereen die aan dit.leerproce s heeft bijgedragen wil ik bij deze hiervoor bedanken. Enkele personen wil ik met name noemen: Mijn promotor, Franz Müller, die mijaltij d de vrijheid heeft gegeven om naast de noodzakelijke ondersteunende werkzaamheden tijd te investeren in zelfstudie en eigen onderzoek. Mijn meest directe collega's:Will y van den Berg,Marti n Bouwmans, Berend Sachteleben, Jillert Santema, Yvonne Soekhram, Jenny Toppenberg, Cees Veeger, LidaVerstege ,Jacque sVervoort , Ton Visser, Gerrit Voordouw, Adrie Westphal en Robert Wijnands. Jullie waren altijd bereid waren om praktische en theoretische problemen op te lossen. Mijn wetenschappelijke vrienden uit Groningen: Jaap Beintema, Jan Drenth, Jan Hofsteenge, Wim Hol,Pete r Jekel, Jan Metske van der Laan, Herman Schreuder, Johan Vereijken, Wicher Weijer en Rik Wierenga. Geweldig, om in zo'n sfeer te mogen samenwerken. Tenslotte Anita, bedankt! CONTENTS ABBREVIATIONS 8 Chapter 1. GENERAL INTRODUCTION 10 1.1 Flavoproteins 10 1.2 Aim of the thesis 12 1.3 Apo flavoproteins 13 1.4 Large scale preparation of apo flavoproteins 17 1.5 FAD-containing disulfide oxidoreductases 19 1.6 Butyryl-CoA dehydrogenase 21 1.7 External flavoprotein monooxygenases 25 1.8 p-Hydroxybenzoate hydroxylase 34 Chapter 2. A study on p-hydroxybenzoate hydroxylase from 61 Pseudomonas fluorescens. A convenient method of preparation and some properties of the apoenzyme. Chapter 3. Large scale preparation and reconstitution of apo 69 flavoproteinswit h special reference to butyryl-CoA dehydrogenase from Megasphaera elsdenii: Hydrophobic-interaction chromatography. Chapter 4. The elucidation of themicroheterogeneit y of highly 81 purified p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens by various biochemical techniques. Chapter 5. Chemical modification of sulfhydryl groups inp - 93 hydroxybenzoate hydroxylase from Pseudomonas fluorescens. Involvement in catalysis and assignment in the sequence. Chapter 6. Chemical modification of tyrosine-38 in p-hydroxy- 105 benzoate hydroxylase from Pseudomonas fluorescens by 5'-p-fluorosulfonylbenzoyladenosine: A probe for the elucidation of the NADPH binding site? Involvement in catalysis,assignmen t in sequence and fitting to the tertiary structure. Chapter 7. The temperature and pH dependence of some properties 117 of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. SUMMARY 125 SAMENVATTING 129 CURRICULUM VITAE 133 ABBREVIATIONS AcPyADP+ - oxidized 3-acetylpyridine adenine dinucleotide phosphate ADP - adenosine 5'-diphosphate 2',5' ADP -adenosin e 2',5'-bisphosphate 2' AMP -adenosin e 2'-monophosphate cDNA - complementary deoxyribonucleic acid CoA(SH) - coenzyme A DTNB - 5,5'-dithio-bis(2-nitrobenzoate) EC -enzym e code EDTA -ethylen e diamine tetraacetate ETF -electro n transferring flavoprotein FAD(H2) - flavin adenine dinucleotide (reduced) FMN - flavin mononucleotide FPLC - fast protein liquid chromatography GSH -glutathion e reduced GSSG -glutathion e oxidized GuHCl -guanidiniu m hydrochloride HPLC - high performance liquid chromatography Kd -dissociatio n constant Km -Michaeli s Menten constant kDa - kilodalton LipS2 - lipoamide oxidized Lip(SH)2 - lipoamide reduced Mr - relative molecular mass NAD(P)+ -nicotinamid e dinucleotide (phosphate) oxidized NAD(P)H -nicotinamid e dinucleotide (phosphate) reduced nm - nanometer NMR -nuclea r magnetic resonance PAGE -Polyacrylamid e gelelectrophoresis p-OHB -par a hydroxybenzoate SDS - sodiumdodecylsulfate SE -sulfoethy l TCA - trichloroacetic acid Parts of chapters 1.3-1.8wil l be published asreviews : la. Methods used to reversibly resolve flavoproteins into the constituents apoflavoprotein and prosthetic group. Muller, F. and Van Berkel,W.J.H . (1989) in:Chemistr y and Biochemistry of Flavoenzymes (Müller, F., ed.)CR CPress , Boca Raton (Florida), inpress . lb. Flavin-dependent hydroxylases with special reference top - hydroxybenzoate hydroxylase. Van Berkel,W.J.H . and Müller, F. (1989) in: Chemistry and Biochemistry of Flavoenzymes (Müller, F., ed.)CR C Press, Boca Raton (Florida), inpress . Chapters 2-7 have been published separately: 2. Müller, F. and Van Berkel,W.J.H . (1982) Eur.J.Biochem. 128, 21-27. 3. Van Berkel,W.J.H. ,Va n den Berg, W.A.M. and Müller, F. (1988) Eur.J.Biochem. 178,197-207 . 4. Van Berkel,W.J.H . andMüller , F. (1987) Eur.J.Biochem. 167,35-46 . 5. Van Berkel,W.J.H. ,Müller , F., Weyer,W.J. , Jekel, P.J. and Beintema, J.J. (1984) Eur.J.Biochem. 145,245-256 . 6. Van Berkel,W.J.H. ,Müller , F., Jekel, P.J., Weyer, W.J., Schreuder H.A. and Wierenga, R.K. (1988) Eur.J.Biochem. 176,449-459 . 7. Van Berkel,W.J.H . and Müller, F. (1989) Eur.J.Biochem. 179,307-314 . Chapter 1 General Introduction 1.1. Flavoproteins Flavoproteins are proteins containing a tightly bound flavin (yellow) molecule usually associated ina non-covalentmode . Flavoproteins are involved in awid e variety of enzymatic reactions spread widely innature . The most occurring flavin prosthetic groups are flavin mononucleotide (FMN)an d flavin adenine dinucleotide (FAD)whic h both can be synthesized fro* riboflavin. Riboflavin (Vitamin B2) is the most abundant flavin com­ pound found innatur e being synthesized by all plants and many microorga­ nisms. The structures of riboflavin, FMN and FAD are shown in Figure 1: Figure 1. Structure of riboflavin (1),FM N (2)an d FAD (3) The isoalloxazine ring system which is the functional part of the fla­ vin prosthetic group can undergo one-o r two-electron transitions which are modulated by the surrounding protein structure. The biological important redox properties of the flavin molecule are outlined in Figure 2: 10 If ? H3C^ H3C^ pKa=K) ftavoquinone ïOrW H3C H3C' FI0J,H 0 Flox© 0 e9 W02 ? f H3Cv H3CN pKa»8.3 fklvosemiquinone H3C' H3C' RH0 oe RH2 0 S e 1«02 f H f e H3Cv. H3C~ pKa=67 ftavohydroquinone » H3C'frV Y H3C'ocijç r FlredH3 Fl^dH20 Figure 2.Redo x states of flavin One of the most fascinating aspects about flavins and flavoproteins is the visible spectral behaviour of the different redox states.A s can be seen from Fig. 2yello w oxidized flavin can be reduced by one electron either to the blue neutral or red anionic semiquinone or reduction can take place by a two-electron process yielding the paleyello w hydroquinone state. Moreover in flavoproteins all kind of
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