Methanol dehydrogenase: A mechanistic study

METHANOL DEHYDROGENASE

A MECHANISTIC STUDY

METHANOL DEHYDROGENASE

A MECHANISTIC STUDY

PROEFSCHRIFT

ter verkrijging van de graad van doctor

aan de Technische Universiteit Delft,

op gezag van de Rector Magnificus,

prof. dr. J.M. Dirken,

in het openbaar te verdedigen

ten overstaan van een commissie aangewezen

door het College van Dekanen

op maandag 6 juni 1988 te 14.00 uur

door

MAARTEN DIJKSTRA ° Promeths-spleinl y

geboren te Zandvoort \% Dci-,-T J$J

Scheikundig ingenieur

Krips Repro Meppel

1988

TRdiss

1635

Prof. dr. ir. A.P.G. Kieboom Prof. dr. ir. J.A.M. de Bont Prof. dr. B.F. van Gelder Prof. dr. O.M. Nèijssel Dr. ir. J.A. Frank Dr. R. Wever

This study was carried out at the Department of Microbiology and Enzymology of the University of Technology Delft, The Netherlands.

Stellingen

1

De spectrofotometrische bepaling van pyridoxal en pyridoxal-5-fosfaat als gekleurd hydrazon na de reactie met phenylhydrazine kan na de ontdekking van PQQ (pyrroloquinoline quinone) als covalent gebonden cofactor van methyl -aminedehydrogenase en amineoxidase niet zonder meer worden toegepast.

- Uada, H. & Snell, E.E. (1961) J.Biol.Chem. 236, 2089-2095

- Van der Heer, R.A., Jongejan, J.A., Frank, J.Jzn. & Duine, J.A. (1986) FEBS Lett. 206, 111-114

2

De aanwezigheid van heterotrofe nitrificeerders in grondmonsters waarin volgens Tate alleen autotrofe organismen verantwoordelijk zijn voor de ni-trificatie, mag op zijn minst opmerkelijk genoemd worden.

- Tate III, R.L. (1980) Appl. Environ. Microbiol. 40, 75-79

3

Het optimisme wat Itoh et al. ten toon spreiden in hun artikel over de

toepassing van PQQ in de oxidatieve decarboxylering van a-aminozuren, zou wel eens getemperd kunnen worden door het ontstaan van redox-inactieve oxazolen uit PQQ.

- Itoh, S., Nobuyuki, K., Ohshiro, Y. & Agawa, T. (1984) Tetrahedron Lett. 25, 4753-4756

- Sleath, P.R., Noar, J.B., Eberlein, G.A. & Bruice, T.C. (1985) J. Am. Chem. Soc. 107, 3328-3338

4

Het uitsluiten van NAD en FAD als cofactor van glucosedehydrogenase in

Acinetobacter calcoaceticus op grond van het ultraviolet absorptiespectrum

van het gezuiverde enzym, zou gezien de vergelijkbare molaire absorptieco­ ëfficiënten van NAD en PQQ bij 260 nm, evenzeer voor PQQ moeten gelden.

De berekening van de redoxpotentiaal van de semichinonvorm van PQQ bij pH 7.3 uit pulsradiolyse metingen volgens Faraggi et al. is, zoals de naam van

het tijdschrift reeds doet vermoeden, een "research" op zichzelf en heeft met "communications" weinig uitstaande.

- Faraggi, M., Chandrasekar, R., Mcwhirter, R.B. & Klapper, H. (1986) Biochem. Biophys. Res. Com. 139, 955-960

6

De bewering van McWhirter & Klapper dat tijdens de oxidatie van substraat door methyl aminedehydrogenase (MADH) uit Bacterium W3A1 de semichinonvorm

van MADH geen katalytische rol speelt, is prematuur gezien de tegenstrijdige resultaten die Kenney & Mclntire met dit enzym hebben gevonden.

- Kenney, U.C., & Mclntire, W.S. (1983) Biochemistry 22, 3858-3868 - McUhirter, R.P. & Klapper, M.H. (1987) in: Flavins & Flavoproteins,

Halter de Gruyter & Co, Berlin, pp. 709-712

7

HOOP doet sneven.

- Woger Onderwijs en Onderzoek Plan (1987), Ministerie van Onderwijs & Wetenschappen, Staatsuitgeverij, 's-Gravenhage.

8

Tijdens s t i l t e s gaan er i n Nederland steeds minder dominees v o o r b i j ; een d u i d e l i j k teken van voortschrijdende s e c u l a r i s a t i e .

9

Voor de natuurwetenschappen blijft er steeds minder natuur over.

10

In de b e s t r i j d i n g van h a r t - en vaatziekten bezorgt de j u i s t e dosering van aspirine menig s p e c i a l i s t hoofdpijn.

"Vertrouwen in grote dingen komt langzaam" OVIDIUS

Aan mijn ouders voor Inge

6 juni, D-Day.

Deze dag zal het einde inluiden van een periode in mijn leven, die be­ heerst werd door PQQ en methanoldehydrogenase. Een periode ook die geleid heeft tot het onstaan van dit proefschrift.

Hoewel slechts één naam de omslag van dit handzame werkje over methanolde-hydrogenase siert, hebben velen binnen en buiten de TU Delft aan het tot stand komen hiervan meegewerkt.

Naast mijn collegae, wetenschappelijk en niet-wetenschappelijk, die ten allen tijden bereid waren hand-en spandiensten te verlenen om mij het werken zo aangenaam mogelijk te maken, wil ik in het bijzonder mijn promotor Hans Duine bedanken voor zijn vele adviezen op het experimentele en literaire vlak; Hans Frank voor zijn vele stimulerende discussies op het stopped-flow gebied; de medewerkers van de werkplaats, in het bijzonder Gerard van der Tooien, die ondanks de vele slapeloze nachten die de anaërobe kast hen onge­ twijfeld bezorgd heeft een adembenemend staaltje van vakmanschap hebben laten zien en Wijnand Schuyl voor zijn ondersteuning bij het gebruik van de laserprinter.

PUBLICATIONS:

The publications designated 1 to 7, which are inserted in this thesis as Chapters II to VIII, respectively, are reprinted by permission from the following Journals:

[1] Dijkstra, M., Van den Tweel, W.J.J., De Bont, J.A.M., Frank, J.Jzn. & Duine, J.A. (1985). Monomeric and dimeric quinoprotein alcohol dehy-drogenase from alcohol-grown Pseudomonas BB1. J. Gen. Microbiol. 131,

3163-3169

[2] Groeneveid, A., Dijkstra, M. & Duine, J.A. (1984). Cyclopropanol in the exploration of bacterial alcohol oxidation. FEMS Microbial. Lett. 25,

311-314

[3] Dijkstra, M., Frank, J.Jzn., Jongejan, J.A. & Duine, J.A. (1984). Inac-tivation of quinoprotein alcohol dehydrogenases with cyclopropane de­ rived suicide substrates. Eur. J. Biochem. 140, 369-373

[4] Frank, J.Jzn., Dijkstra, M., Duine, J.A. & Balny, C. (1988). Kinetic and spectral studies of the redox forms of methanol dehydrogenase from

Hyphomicrobium X . Eur. J. Biochem., in the press

[5] Dijkstra, M., Frank, J.Jzn., Van Wielink, J.E. & Duine, J.A. (1988). The soluble cytochromes of methanol-grown Hyphomicrobium X: evidence

against the involvement of autoreduction in electron acceptor func­ tioning of cytochrome c.. Biochem. J. 251, 467-474, copyright (c) The

Biochemical Society, London

[6] Dijkstra, M., Frank, J.Jzn. & Duine, J.A. (1988). Studies on electron transfer from methanol dehydrogenase to cytochrome c , both purified

from Hyphomicrobium X. Biochem. J., accepted for publication

[7] Dijkstra, M., Frank, J.Jzn. & Duine, J.A. (1988). Methanol oxidation under physiological conditions using methanol dehydrogenase and a fac­ tor isolated from Hyphomicrobium X. FEBS Lett. 227, 198-202

[8] Duine, J.A., Frank, J.Jzn., Jongejan, J.A. & Dijkstra, M. (1984). En-zymology of the bacterial methanol oxidation step. In: Microbial Growth

(Crawford, R.L. & Hanson, R.S., eds.) pp. 91-96

[10] Duine, J.A., Frank, J.Jzn. & Dijkstra, M. (1987). Quinoproteins in the dissimilation of C -compounds. In: Microbial Growth on C -compounds. Proceedings of the 5th International Symposium. (Van Verseveld, H.W. & Duine, J.A., eds.) pp. 105-112

[11] Dijkstra, M., Frank, J.Jzn. & Duine, J.A. (1985). Mechanistic studies on methanol dehydrogenase. Anthonie van Leeuwenhoek 51, 443

The Figures 6, 7 and 8 in Chapter I are reprinted by permission from the following Journals:

- Mincey, T., Bell, J.A., Mildvan, A.S. & Abeles, R.H. (1981). Mechanism of action of methoxatin-dependent alcohol dehydrogenase. Biochemistry 20,

7502-7509

- Parkes, C.H. & Abeles, R.H. (1984). Studies on the mechanism of action of methoxatin-requiring methanol dehydrogenase: reaction of enzyme with elec­ tron acceptor dye

- 0'Keeffe, D.T. & Anthony, C. (1980). The interaction between methanol de­ hydrogenase and the autoreducible cytochromes c of the facultative meth-ylotroph Pseudomonas AMI. Biochem. J. 190, 481-482, copyright (c), The

Contents

Abbreviations 10

Chapter I General introduction 11

Chapter II < Monomeric and dimeric quinoprotein alcohol

dehydrogenase from alcohol-grown Pseudomonas BB1 35

Chapter III Cyclopropanol in the exploration of bacterial

alcohol oxidation 51

Chapter IV Inactivation of quinoprotein alcohol dehydrogenase

with cyclopropane-derived suicide substrates 59

Chapter V Kinetic and spectral studies on the redox forms of

methanol dehydrogenase from Hyphomicrobiurn X 75

Chapter VI The soluble cytochromes c of methanol-grown

Hyphomicrobiurn X: evidence against the involvement

of autoreduction in electron acceptor functioning of

cytochrome c 97

Chapter VII Studies on electron transfer from methanol dehydrogenase to cytochrome c , both purified

from Hyphomicrobiurn X 117

Chapter VIII Methanol oxidation under physiological conditions using methanol dehydrogenase and a factor isolated

from Hyphomicrobiurn X 139

ABBREVIATIONS

PQQ pyrroloquinoline quinone, semi systematic name for 2,7,9-tricarboxy-lW-pyrrolo(2,3-f)quinoline-4,5-dione PQQH' semiquinone form of PQQ

PQQH quinol form of PQQ

Wurster's blue free radical of N,N,N',N'-tetramethyl-p-phenylene-diamine

PMS phenazine methosulphate MDH methanol dehydrogenase MDH . fully reduced form of MDH

red

MDH , MDH . semiquinone form of MDH sem ^.oxl

MDH , MDH fully oxidized form of MDH ox ox J

MDH .S, MDH . complex of MDH with substrate ox ox2 ox MDH. MDH (7-n vivo)

IV

Mops 4-morpholinepropanesulphonic acid

Hepes 4-(2-hydroxyethyl)-l-piperazine-ethanesuTphonic acid Tri cine N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]-glycine Tris 2-amino-2-hydroxymethylpropane-l,3-diol

Ches 2-(N-cyclohexylamino)ethanesulphonic acid SDS sodium dodcyl sulphate

PAGE polyacrylamide gel electrophoresis M molecular mass

r

ESR electron spin resonance

HPLC high performance liquid chromatography THF tetrahydrofolate

EDTA ethylenediaminetetra-acetate GSH reduced gluthathione

ATP adenosine 5'-triphosphate E' midpoint potential at pH 7.0 K Michaelis constant

m

11

-Chapter I

General introduction

1. METHYLOTROPHIC BACTERIA

The major sources of methanol production in nature are the oxidation of methane and the hydrolysis of methylester moieties present in pectin and lignin, the principal structural components of plants. The methanol produced can serve as an energy and carbon source for the so-called methylotrophic bacteria. Obligate methylotrophs grow only on C -compounds (that is com­ pounds containing no carbon-carbon bonds) like methane, methanol, methylated amines or formate, whereas facultative methylotrophs are also able to uti­ lize multicarbon substances. During our research on methanol dehydrogenase (MDH) two methylotrophic bacteria have been predominantly used, namely Hyphomicrobium X and Pseudomonas BB1.

Hyphomicrobium X

Hyphomicrobium species belong to the prostecate bacteria which reproduce

by a budding process [2-4]. They are Gram-negative, stalked, motile bacteria (0.5 -1.0 x 1.0 -3.0 /jm), which are rod-shaped with pointed ends, having hyphae (prostheca or stalks) from which a daughter cell develops asymmetri­ cally [1]. All strains grow with 0? as an electron acceptor, some are capa­

ble of growing anaerobically with nitrate as an electron acceptor [5,6]. The optimum growth temperature lies between 2 5 - 3 7 C at neutral or slightly alkaline pH. They are restricted facultative methylotrophs, because besides reduced C - compounds they can utilize some multicarbon compounds like acetate, ethanol and 3-hydroxybutyrate, which are metabolized via conversion to acetyl-CoA. Hyphomicrobium X was first isolated by Attwood & Harder [1]

from enrichment cultures obtained under denitrification conditions in the presence of methanol. Methanol is oxidized to formate and further dissimi­ lation occurs in a cyclic way in the so-called serine pathway and in a

direct way to formaldehyde and eventually to CO and H O [7]. Strains have been isolated which grow on dichloromethane, dimethylsulphoxide or dimethyl-sulphide [8,9]. In the absence of a carbon source in the culture medium, sometimes slow growth is observed at the expense of carbon compounds from the atmosphere (oligocarbophilic growth).

Pseudomonas BBl

Pseudomonas species which grow on methanol are Gram-negative, rod-shaped

cells with a single polar flagellum. They are catalase- and oxidase positive and grow strict aerobically on a number of C -compounds and multicarbon substrates [7]. Methanol is assimilated via the serine pathway. Pseudomonas

BBl (1.0-1.5 x 2.5-4.0 /im) was first isolated by De Bont et al. [10] from

enrichment cultures on the acetyl.enic compound 3-butyn-l-ol. It contains a red pigment and inclusions of reserve material, probably poly-3-hydroxy-butyrate. Besides 3-butyn-l-ol, Pseudomonas BBl can grow on several alcohols

like methanol, ethanol, 1-propanol and 1,2-propanediol, but also on a variety of other multicarbon compounds like acetate, malonate and succi-nate [10].

2. QUINOPROTEIN ALCOHOL DEHYDROGENASES

In the past it has become clear that bacterial oxidation of alcohols to aldehydes is not only catalysed by NAD(P)-dependent alcohol dehydrogenases but can also proceed via a NAD(P)-independent, dye-linked alcohol dehydro-genase. The latter enzyme, active in Gram-negative methylotrophic bacteria, is called methanol dehydrogenase (MDH, EC 1.1.99.8). The cofactor in this enzyme appears to be a novel one, pyrroloquinoline quinone (PQQ), and all bacterial dye-linked alcohol dehydrogenases characterized sofar are quino-proteins (PQQ-containing enzymes). They differ however from each other in substrate specificity and several other properties [11]. Since they can be regarded as separate entities, the enzymes are indicated with a name related to an alcohol which is a good substrate for the organism from which the

13

-Table 1. Quinoprotein alcohol dehydrogenases

Name Organisms References

Methanol dehydrogenases MDH NAD+/PQQ dependent MDH Gram-negative methylotrophs [7] Gram-positive methylotrophs [23] Ethanol dehydrogenases Quinoprotein ethanol dehydrogenase Quinohaemoprotein ethanol dehydrogenase Pseudomonas aeruginosa [26] Pseudomonas putida, biovar.B [a]

Pseudomonas testosteroni [27]

Acetic acid bacteria? [28,29]

Poly alcohol dehydrogenases

Glycerol dehydrogenase Polyvinylalcohol dehydrogenase Polyethyleneglycol 6000 dehydrogenase Quinate dehydrogenase Gluconobacter industrius Synergystic culture of Pseudomonas sp.VM15C and Pseudomonas putida VM15A

Synergystic culture of Flavobacterium and Pseudomonas species Pseudomonas aureofaciens, Acinetobacter calcoaceticus [30] [31] [32] [b]

a) Van der Meer, R.A. & Groen, B.W., unpublished results. b) Van Kleef, M.A.G., unpublished results.

COOH HN /COOH HOOC P Q Q COOH HN /COOH HOOC PQQH' p y r r o l o - q u i n o t i n e p y r r o l o - q u i n o l t n * quinone semiquinon» HN /COOH OH HOOC PQQH2 pyrrolo - q u i n o l i n * quinot

Fig.l. Structure of PQQ, PQQH' and PQQH

PQQ, pyrroloquinoline quinone; PQQH', semiquinone form of PQQ; PQQH , quinol form of PQQ

enzyme studied was isolated (Table 1 ) . The properties of PQQ and of each enzyme are briefly discussed below.

Discovery and some properties of PQQ

Although MDH from Pseudomonas M27 was already purified and characterized

in 1965 by Anthony & Zatman [12] and its unusual cofactor already indicated, it took nearly 15 years before the structure of the cofactor was elucidated. Originally a pteridine [13] and later on a lumazine derivative [14] was pro­ posed to be the cofactor. Subsequently it was shown that it was a nitrogen containing o-quinone [15]. Eventually the chemical structure of the cofactor was elucidated independently by the groups of Duine et al. [16] and

Salisbury et al. [17]. It appeared to be

2,7,9-tricarboxy-lW-pyrrolo(2,3f)-quinoline-4,5-dione (Fig.l.). The latter research group proposed the name methoxatin and Duine et al. the semi systematic name pyrroloquinoline qui­

none, abbreviated to PQQ (Fig.l). Enzymes containing PQQ were indicated as quinoproteins. Three redox forms of PQQ participate in biological oxida­ tions: PQQ, PQQH' and PQQH (Fig.l, section 3 ) . The absorption spectra of PQQ and its quinol form (PQQH ) are shown in Fig.2. PQQH in a buffer of pH 7.0 has an absorption maximum at 302 nm and it does not fluoresce [18]. Since PQQ is partly hydrated in water [19], the absorption spectrum of "PQQ" is the sum of the contributions of PQQ and PQQ-HJ) [19] and the fluorescence

15 -1.4 1.2 S 1.0 C feO.8 t/5 JU * 0.6 0.4 0.2 0 -'"' ƒ 1 ' ' / I I ' / : \ / \ i / 1 \ \ 1 l \ AN \ \ i i r -1.4 1.2 1.0 0.8 0.6 0.4 0.2 200 300 4 0 0 500 Wavelength (nm) 600 700

Fig.2. Absorption spectra of PQQ and PQQH

PQQ was dissolved in 0.05 M-potassium phosphate pH 7.0 and the absorption spectrum of the solution measured before (- - -) and after reduction ( ) with H in the presence of Pt02.

of such solutions originates from PQQ-^O [19,20]. The absorption spectrum of PQQH' is not exactly known since it is in equilibrium with PQQ and PQQH2

-The spectrum obtained after mixing of equimolar concentrations of PQQ and PQQH under anaerobic conditions in the presence of Li -ions showed absorp­ tion maxima at 340 and 458 nm as well as a broad band at 600 nm [18]. Sub­ stituting Li+ for Na- or Cs-ions, shifted the broad absorption band at 600

nm to higher wavelenghts with a concomitant decrease of the ESR signal and a disappearence of the hyperfine structure in the signal. This is indicative for the formation of a diamagnetic complex between an alkali metal ion and two PQQH''s [18]. MDH , the enzyme form as it is isolated, contains PQQH', as is evident from ESR spectra [21]. This enzyme form has a complicated absorption spectrum between 300 and 450 nm (suggesting overlapping absorp­ tion bands), a shoulder at 475 nm and also a very broad band at 600 nm. Semiquantitative free radical measurements showed that the cofactor is not

Hu_ XOOH

HOOD

Scheme 3 R=C„H2„„ " \NH2

Fig.3 Adducts of amines and NH with PQQ

completely in the free radical form (see also [22]). These observations suggested that similar equilibria, as observed for the model system [20], might exist in MDH , according to:

3 oxl 3

2 PQQH" PQQ + PQQH, (PQQ + PQQH )complex.

However, as will be shown in Chapter II, the similarity in ESR- and absorp­ tion spectra of monomeric and dimeric MDH of Pseudomonas BB1 argues

against such an explanation. As to the chemical reactivity of PQQ, it was shown that the C:5 carbonylgroup of PQQ is very reactive towards

nucleo-philic agents like aldehydes, ketones, alcohols and amines, leading to adducts with a more or less similar absorption spectrum [19,20]. Although the adducts of NH with PQQ (Fig.3) seem relevant since NH -salts are nec­ essary as an activator in the MDH assay (see pp.18 and Chapter V) evidence for such a relationship is not available and there are even contraindica­ tions. For instance, addition of NH does not change the ESR signal of MDH ,, whereas a significant change is observed when NH, is added to a

so-oxl 3 lution of PQQH' [20]. Therefore the role of NH could also be explained from

an interaction with the protein part of the enzyme or with a different moiety of the PQQ molecule, for instance the carboxylgroup at the C-9 posi­ tion [20].

NAD-dependent PQQ-containing methanol dehydrogenase

This enzyme, present in a multienzyme complex with NAD -dependent aldehyde dehydrogenase and NADH-dehydrogenase, was found in Nocardia sp.239 [23], a

17

-Gram-positive methylotroph, capable of growing on methanol and in Methylo-coccus capsulatus, strain Bath, a Gram-negative methanotroph [23]. Just like

classical MDH it contains PQQ. Activity can only be measured in the presence of NAD , artificial electron acceptor dyes and NH -salts as activator. The activator dependency develops during exposure of the complex to 0 , a pro­

perty which has occasionally been found for MDH as well [24]. Compared to MDH the substrate specificity is very restricted, only methanol being a sub­ strate. Cyclopropanol, an irreversible inhibitor of MDH (see pp.19), does not inhibit the NAD-dependent, PQQ-containing enzyme.

Quinoprotein ethanol dehydrogenase

Some alcohol- or alkane-grown, non-methylotrophic bacteria contain a NAD(P)-independent, dye-linked alcohol dehydrogenase. Such an enzyme was originally purified from a bacterium [25], which has meanwhile been classi­ fied as a Pseudomonas putida, biovariant B strain (Van der Meer, R.A. &

Groen, B.W., unpublished results). An other type of this enzyme is present in Pseudomonas aeruginosa [26]. The enzyme from the latter organism is a

monomer with a M of 101 000, containing 2 molecules of PQQ per enzyme mole-r

cule. Cyclopropanol is a suicide substrate for the enzyme, but only 1 mole­ cule is necessary for complete inactivation [26]. In contrast to MDH it oxi­ dizes not only primary alcohols (with the exception of methanol) and form­ aldehyde but also secondary alcohols and higher aldehydes. Whereas NH. -salts are poor, higher alifatic amines are excellent activators.

Quinohaemoprotein ethanol dehydrogenase

Addition of PQQ to a cell-free extract of Pseudomonas testosteroni, grown

on alcohols like ethanol, propanol and butanol [27], induces dye-linked alcohol dehydrogenase activity. In contrast to MDH, the pH optimum was lower (pH 7.7) and NH -salts or amines were not required as activators. Ca -ions however, were essential for PQQ-binding and activity. The enzyme was a mono­ mer (M 60 000) containing 1' haem c group. Full reconstitution of the apo-enzyme required 1 molecule of PQQ per apo-enzyme molecule [27]. With the

excep-tion of methanol, primary and secondary alcohols and aldehydes were sub­ strates. Both one and two electron acceptors (anionic and cationic) were active: This enzyme is probably also present in Acetobacter and Glucono-bacter species [28,29] in its holoenzyme form.

Polyalcohol dehydrogenases

Glycerol dehydrogenase [30], polyvinylalcohol dehydrogenase [31] and poly-ethyl eneglycol 6000 dehydrogenase [32] are quinoprotein polyalcohol dehydro­ genases. The last two enzymes are apoenzymes. As a consequence degradation of the substrates only takes place in synergystic cultures in which PQQ is supplemented by another organism. Quinate dehydrogenase has been detected in

Acinetobacter and Pseudomonas species (Van Kleef, M.A.G., unpublished

results).

MDH (EC 1.1.99.8)

As already mentioned, this enzyme is present in all Gram-negative bacteria capable of utilizing methane and methanol and constitutes about 10 - 20 % of the soluble protein in the cell [7]. The enzyme normally consists of two probably identical subunits and contains 2 molecules of PQQ. The subunits have M values of approx. 60 000, the M values of the native proteins are in the range of 120 000 to 158 000. Exceptions are the enzymes from Hethylo-monas methanica [33] and Methylosinus sporium [34], which are monomers with M values of about 60 000. MDH catalyzes the oxidation of primary alcohols and formaldehyde.

MDH is assayed at pH 9.0 or higher in the presence of NH -salts as acti­ vator and a cationic artificial electron acceptor like phenazine metho-sulphate (PMS) or Wurster's blue [19].

Preliminary investigations on the MDH of Pseudomonas BB1 indicated that

monomeric as well as dimeric MDH is present. Since controversial views on the mechanism of MDH existed at that time, but monomeric and dimeric MDH from different organisms were used in these studies, it seemed worthwhile to further investigate this observation. Curiously, it appeared that

Pseudo 19 Pseudo

-monas BB1 contains monomeric as well as dimeric MDH, the ratio depending on

the growth conditions (Chapter II). Purification showed that both enzyme forms resemble in many respects MDH from other methylotrophs, although their substrate specificity lies in between that of MDH and ethanol dehydrogenase. A kinetic study revealed that the parameters of both enzyme forms were al­ most the same, indicating that dimerization did not influence the catalytic properties of the subunits. Therefore, it seems highly unlikely that the controversion on the mechanism of MDH could be related to the monomeric or

dimeric nature of the enzyme. Since monomeric enzyme contained one molecule of PQQ per enzyme molecule, it is not likely that the two prosthetic groups in the dimeric enzyme interacted which each other. Monomeric and dimeric enzyme could not be converted into each other. Extraction of the cyclo-propanol-inactivated quinoprotein alcohol dehydrogenases from whole cells of

Pseudomonas BB1 showed that both enzyme forms probably are functional in

alcohol oxidation in vivo (Chapter II, III and IV).

MDH activity in whole cells is inhibited by EDTA, phenylhydrazines and high phosphate concentrations [35,36]. Cyanide and hydroxylamine are compe­ titive inhibitors for substrate [37] or activator [38] in the assay. Recent­ ly Mincey et al. introduced cyclopropanol as an irreversible inhibitor in

their studies to elucidate the redox cycle of MDH [22]. Since we found that the inhibition was due to the modification of PQQ (Chapter IV), it was temp­ ting to investigate the effect on other quinoproteins. Application of cyclo­ propanol and cyclopropane-derived substrates on whole.cells.of several bacteria revealed that these compounds selectively inhibit quinoprotein alcohol dehydrogenases (Chapter III). Inhibition of m and ethanol-grown Hyphomicrobium X showed that methanol and ethanol oxidation is com­

pletely blocked and formaldehyde oxidation is not. This indicates that ethanol oxidation of cells grown under these conditions occurs via MDH. Formaldehyde oxidation could either partially proceed via MDH or exclusively via other enzymes such as a NAD-dependent aldehyde dehydrogenase (Poels, P., unpublished results) or a dye-linked aldehyde dehydrogenase [39]. These ex­ periments also showed that some bacteria contained both a quinoprotein as

O . 2 ÜJ CJ Z <

m

er

o

m

WAVELENGTH (nm)

Fig.4. Absorption spectra of HOH Spectra of MDH(red)( ) ; MDH(oxl)(-measured in 0.05 M-Mops buffer pH 7.0

■); MDH(ox2)( ) were

well as an NAD-dependent alcohol dehydrogenase, the latter being insensitive for these compounds. In the case of Pseudomonas aeruginosa, it appeared that

ethanol oxidation proceeds via both enzymes to the same extent (Chapter III). Therefore, cyclopropanol is an excellent probe to study the physio­ logical role of certain quinoprotein alcohol dehydrogenases in the bacterial cell.

3. THE IN VITRO CYCLE OF HDH

MDH is usually isolated in a form which is unable to react with substrate [21]. As already mentioned this form contains PQQH' and is designated M D HQ X 1

(Fig.4). Protecting the enzyme with cyanide or hydroxylamine against inacti-vation by excess electron acceptor in the presence of activator, a stable

21 -( P O O H ' ) -( P O Q ) M O HO X ) 1 - e l e c t r o n a c c e p t o r M D H0 X i s o l a t e d e n z y m * ■> ' - e l e c t r o n donor ( P Q Q H j ) - » MOH red r e d u c e d form l a b i l e , o x i d i z e d f o r m CN" r M O H0,? s u b s t r a t e

Fig.5. The in vitro reaction cycle of dimeric MDH as proposed by Duine et al. [40]

The different redox forms of the enzymes are indicated by abbreviations, which are explained in the text.

oxidized form of MDH (MDH , Fig.4) was produced. MDH could in turn be converted by substrate into a fully reduced form of MDH (MDH ,, Fig.4) with concomitant product formation [37]. Extraction of MDH showed that it con­ tained PQQH? [40]. Titration of MDH . with electron acceptor in the

presence of activator produced another fully oxidized intermediate of MDH (MDH ) which was very labile. It could be converted with cyanide into MDH „ [40]. MDH , can be reduced to MDH . with a one electron donor

ox2 L oxl red

(methyl viol ogen, [41]), thereby confirming the intermediate position of *

MDH , between the fully oxidized forms, MDH and MDH „, and the fully oxl ox ox2

reduced form, MDH ., as originally proposed by Duine et a7. [40] and de­

picted in Fig.5.

Shortly afterwards Mincey et al. [22] postulated a quite different mecha­

nism for monomeric MDH from Methylomonas methanica (Fig.6). In their view,

only enzyme molecules containing PQQH' are active, leading to a 3-electron reduced form of PQQ after substrate conversion. Product release only occurs after oxidation of the enzyme-substrate complex by electron acceptor. Since their main arguments were derived from results of experiments with the sui­ cide substrate cyclopropanol and ESR measurements [22], similar studies were

E ox P Q Q H ' " ^ ox PQQH'

V

v

Fig.6. The in vitro reaction cycle of monomeric MDH according to Mincey

et al. [22].

Abbreviations used: E(ox), oxidized enzyme, identical to MDH(oxl); E(red), reduced enzyme, identical to MDH(red); A(ox) and A(red) oxidized and re­ duced forms of an electron acceptor respectively; S, substrate; P, product.

started with MDH from Hyphomicrobi'urn X. It appeared soon that Mincey et al.

had misinterpreted their ESR spectra [41]. Moreover, our results on inhibi­ tion of monomeric and dimeric enzyme from Pseudomonas BBl and dimeric enzyme

from Hyphomicrobiurn X (Chapter IV) with cyclopropanol were quite different.

In our case, cyclopropanol behaved as a normal substrate since it only re-*

acted with MDH and not with MDH , or MDH .. Therefore, the arguments for ox oxl red

the view of Mincey et al. [22] that only enzyme molecules containing PQQH"

are catalytically active could be refuted.

The mechanism of inactivation of quinoprotein alcohol dehydrogenases by cyclopropanol and cyclopropanone hydrate probably proceeds via a ring open­ ing such as proposed for the metal ion catalysed degradation of cyclopropane

23

-E.A .S

E . A .CN _{E A}o . A _ O H

Fig.7. Another reaction cycle of monomeric MDH, as proposed by Parkes & Abeles [44]

Abbreviations used: E', oxidized enzyme, identical to MDH(oxl); E, reduced enzyme, identical to MDH(red); A(ox) and A(red), oxidized and reduced e-lectron acceptor dye respectively; E-A(ox), enzyme-dye complex, identical to MDH(ox), E-A(ox)-CN, enzyme-dye-cyanide complex, identical to MDH(ox2); E-A(ox)-A-0H, enzyme-dyg-cyclopropanol complex, identical to

cyclopro-panol-inactivated MDH(ox); S, substrate; P, product.

derivatives [42,43], leading to ^-propionaldehyde or /3-propionic acid ad-ducts of PQQ at the C-5 position, respectively (Chapter IV). The proposed structures have meanwhile been proven (Frank, J.Jzn., unpublished results).

In 1984, Parkes & Abeles [44] reported further mechanistic studies on mo-nomeric MDH from Methylomonas methanica (Fig.7]). Curiously, it was pro­

posed that the conversion of MDH into MDH , at high pH (E'and E in Fig. 7) involved no electron transfer, the presence of electron acceptor dye only accelerating this step. This is in disagreement with our observations that MDH , and MDH . can only be converted into each other using one electron

oxl red

acceptors [37] or one electron donors [41]. Furthermore, we had already found that the conversion of MDH . into MDH . with different dyes, yields

preparations with identical absorption spectra. It was also claimed [44] *

that MDH is a dye-enzyme complex, formed in a reaction which does not lead to dye reduction. This complex can participate in several reactions. For in­ stance, addition of substrate yields a MDH-dye-substrate complex which gives rise to dye reduction and product formation (Fig.7). Although we reject the existence of such a complex on the afore mentioned grounds, the idea that MDH has first to react with electron acceptor is in agreement with our view that enzyme molecules in the semiquinone state are not catalytically active. Another reaction of this complex is the reversible addition of cyanide, leading to a MDH-dye-cyanide complex (corresponding to MDH ) . This is also difficult to reconcile with the fact that identical spectra of MDH „ were

ox2 obtained after oxidation of MDH . with different electron acceptors [37].

In Chapter V evidence is presented that the three redox forms in the cata-*

lytic cycle of MDH (MDH . , MDH , and MDH ) are genuine redox forms of

J v oxl red ox ' 3

MDH and not enzyme-dye complexes as proposed by Parkes & Abeles [44]. The stoichiometry of the oxidation of MDH . to MDH , and MDH , to MDH by

red oxl oxl ox Wurster's blue (Chapter V) is in agreement with the postulated redox states of the cofactor in the enzyme forms ([40], Fig.4). It is further shown that in the presence of substrate, MDH is not detected because it is rapidly converted into an enzyme-substrate complex (MDH .S).

On studying the individual steps of the catalytic cycle with stopped-flow spectrophotometry, it appeared that the oxidation of the reduced forms of MDH by Wurster's blue occurred much faster at pH 9.0 than at pH 7.0. At pH 9.0 the rate-limiting step in the catalytic cycle (in the absence of acti­ vator) is the conversion of the oxidized enzyme-substrate complex (MDH .S)

2

into MDH . and product. Under these conditions C H OH gave an isotope ef­ fect of 7. The effect of the activator was confined to this step, decreas­ ing the isotope effect to 1.4 at saturating conditions, indicating that the substrate oxidation rate approximated the oxidation rate of reduced MDH. Making use of the large isotope effect in the absence of activator, the

2

transient MDH .C H_OH complex could be isolated and its decay into MDH .

25

-and formaldehyde demonstrated. All these results are in agreement with the original view on the redox cycle of MDH as proposed by Duine & Frank [40].

Biological oxidations can either proceed via a hydride transfer or a radi­ cal mechanism. Maclnnes et al. [45] used cyclopropane-derived compounds like

cyclopropylmethanol to support the view that alcohol oxidation by NAD-de-pendent alcohol dehydrogenases occurs via hydride transfer. However, Sherry & Abeles showed that the flavoprotein methanol oxidase from Hansenul a poly-morpha is irreversibly inactivated by cyclopropanol via a radical mechanism

[46]. Although we assume that the inactivation of MDH by cyclopropane-de­ rived substrates proceeds via a radical mechanism (Chapter IV), this does not implicate that the oxidation of genuine substrates also occurs in this way. The large isotope effect (Chapter V and VII) of the substrate oxidation step is indeed in accordance with hydride transfer. On the other hand it is clear that the oxidation of MDH , and MDH , proceeds via one electron

red oxl

steps and that the role of activator is confined to the substrate oxidation step (Chapter V ) .

4. ELECTRON TRANSFER FROM NDH TO THE RESPIRATORY CHAIN

At first sight the electron transport chain of methylotrophic bacteria is similar to that of other bacteria since it consists of ubiquinones, iron-sulphur proteins, midchain b-type cytochromes, c-type cytochromes and cy­ tochrome oxidases [47]. However, whereas dehydrogenases oxidizing organic substances other than methanol are coupled to the respiratory chain prior to cytochrome b, MDH appears to donate its electrons to the electron transport chain at the level of cytochrome c [7] and the number of cytochromes c in­ creases at growth on methanol. The assumption that MDH donates its electrons to cytochrome c seems plausible since mutants of Methyl obacten'urn sp. strain

AMI lacking cytochrome c were unable to grow on methanol [48], cytochrome c reduction by methanol in membrane vesicles of Methylobacten'um sp.strain AMI

was not inhibited by antimycine A, a known inhibitor of cytochrome b oxida­ tion [49] and the P/0 ratio (the amount of ATP produced with the transport

of 2 electrons to oxygen) with methanol as a substrate is less compared to that of succinate or NADH [50]. Both MDH and the soluble cytochromes c are localized in the same compartment of the cell, the periplasmic space [51-53].

The soluble cytochromes c of methylotrophic bacteria

The major soluble cytochromes c found in methylotrophic bacteria are cy-tochrome c and cycy-tochrome c , a subdivision according to the isoelectric points (pi) and redox potentials (E') [47], cytochrome c having the lowest values. In other respects the cytochromes are similar; both are monomeric proteins and contain one haem c group, having histidine and methionine as the axial ligands and showing a characteristic absorption band at 550 nm. The role of cytochrome c as primary electron acceptor for MDH was origi­ nally derived from in vitro experiments [54,55]. Recently Nunn & Lidstrom

showed that mutants from Methylobacteriurn sp.strain AMI, lacking cytochrome

c. , but not cytochrome cu or MDH, were unable to grow on methanol [56,57]. L n

This suggests that in vivo, cytochrome c also accepts electrons from MDH

while cytochrome cu probably accepts electrons from cytochrome c. and

trans-n L fers them to its oxidase [47].

Both cytochromes c. and c„ from Methylobacteriurn sp.strain AMI and M. methylotrophus have a peculiar property. In the absence of added reductant

reduction of the ferricytochromes c is observed at high pH, the so-called autoreduction [54,58]. In the presence of MDH it has been claimed that auto­ reduction occurs at much lower (physiological) pH values (in the case of

Acetobacter methanolicus even at pH 4.0 [54,59,60]) probably by lowering the

dissociation constant of the electron donorgroup (Fig.8). For that reason it was suggested by Anthony & coworkers [58,61] that autoreduction plays a role in the electron transfer mechanism between MDH and cytochrome c. . Assuming that autoreduction is also involved in the in vivo process, then the radical

species {(3) in Fig.8} is oxidized by cytochrome c oxidase {to species (4)} which in turn is reduced by MDH {to species (2)}.

27 -(1) H * (2) (3) F e3 + PKA> 1 0 s F o ^+ autoreduction Fp^+ • XH □K is less than 8.5 A in the presence ol MDH e — * • (ferricyanide)

Fig.8. Autoreduction mechanism of cytochrome c and its involvement in the reaction with MDH according to 0'Keeffe & Anthony [58]

The electron donor in the autoreduction process is a weakly acidic group (XH) dissociating at high pH values or in the presence of MDH at physio­ logical pH, providing a negatively charged species which is able to donate an electron to the haem. (1), ferricytochrome c(L); (2), the deprotonated form of (1) at high pH or in the presence of MDH; (3), radical complex of ferrocytochrome c(L); isoelectronic with species (2); (4), radical complex of ferricytochrome c(L), which is not autoreducible but reducible by MDH.

In Chapter VI it is shown that Hyphomicrobium X, grown on methanol, also

contains a cytochrome c. and a cytochrome c . Both were monomeric proteins L H

containing 1 haem c group per protein molecule. Native cytochrome c has a M value of 19 500, a pi value of 4.3 and an E' value of +270 mV. The values

r o for cytochrome cu were 15 000, 7.4-7.5 and +290 mV respectively. Both

cyto-H

chromes also showed autoreduction at high pH, although with a much lower rate compared to the cytochromes c from other methylotrophic bacteria.

Cytochrome c appeared indeed to be the natural electron acceptor for MDH. It was instantaneously reduced by MDH . or MDH , at pH 7.0, but much

red oxl

slower at pH 9.0, whereas the reduction rate of cytochrome cu was 7-fold n

lower and did not vary with the pH. The soluble cytochromes c of other or­

ganisms show an opposite behaviour with respect to the pH. Not only much lower reduction rates but also different pH optima have been reported [54, 55]. Since we observed no reduction of cytochrome c on addition of

respon-sible for the stimulation of the autoreduction process in the case of Hypho-microbium X. However, it cannot completely "be excluded that cyclopropanol

inactivated enzyme is an unsuited conformation, preventing it to bind in the appropriate way to cytochrome c .

Functional coupling of MDH to cytochrome c

Although it is obvious that MDH donates its electrons to cytochrome c in the electron transport chain [7], methanol-dependent cytochrome c reduction could not be attained in vitro at high rates. Since permeabilised cells were

able to oxidize methanol, a property which was lost after exposure to oxygen [24], the possibility was considered that the components became inactivated during the aerobic isolation and purification. Since a complex containing cytochrome c and MDH, isolated under anaerobic conditions, showed indeed methanol-dependent cytochrome c reduction this hypothesis seemed valid [24]. After exposure to 0 , cytochrome c could no longer be reduced by the addi­ tion of methanol. Moreover, MDH was changed by 0 since a free radical was induced in the enzyme together with a change in the absorption spectrum, characteristic for the presence of the semiquinone form of MDH (MDH ) and MDH activity in the dye-linked assay could no longer be detected without the presence of activator [24]. To explain the development of the activator-de­ pendency it has been proposed that 0 transforms MDH into a different form, in which e.g. a lysyl residue acting as an internal activator in vivo is no

longer active [40,62], making it therefore dependable on an externally pro­ vided activator like NH. -salts.

4

In contrast with this observation, other reports showed that methanol-de­ pendent cytochrome c reduction occured under aerobic conditions using the purified components from Methylomonas J [55], Hethylophilus methylotrophus

or Methyl'obacteri'urn sp.strain AMI [54], the reaction monitored by using an

excess of horse heart ferricytochrome c as final electron acceptor. The turnover rates which could be attained were, however, extremely low compared to the electron transport rates in whole cells (less than 1 %) and the arti­

be 29 be

-tween MDH and cytochrome c . Since this is in contradiction with our obser­ vation that an immediate reaction occurs between MDH . and ferricytochrome c from Hyphomicrobium X (Chapter VI) it was decided to study the individual

steps of the redox cycle of MDH with ferricytochrome c as electron acceptor (Chapter VII).

Comparison of the catalytic cycles of MDH with Wurster's blue and cyto­ chrome c as electron acceptor reveals that the dissimilarities between the electron acceptors can be explained from different rate-limiting steps in the reaction cycles. Ferricytochrome c is an excellent oxidator of reduced MDH at pH 7.0. At this pH the substrate oxidation step is slow and NH.C1 is a poor activator. However, at pH 9.0 ferricytochrome c is a poor oxidator of MDH , and MDH and since no isotope or activator effect was observed

red sem

under these conditions, substrate oxidation is not rate-limiting. As already mentioned the opposite holds for the artificial dye system. At pH 9.0, Wurster's blue is an excellent oxidator of MDH . and MDH the substrate

red sem oxidation step being rate-limiting although activator can relieve this. These differences in the nature of the rate-limiting step explain the insig­ nificant stimulating effects of activator and the different pH optimum for the ferricytochrome c system. It also explains why ferricytochrome c. is a poor electron acceptor in an assay for MDH since the substrate oxidation step is rate-limiting and NH Cl is an inadequate activator at this pH. The low turnover rates observed by others [54,55] in the MDH/ferricytochrome c

system are most probably related to a rate-limiting electron transfer be­ tween ferrocytochrome c and horse heart ferricytochrome c. Since EDTA and high salt concentrations inhibit the oxidation of MDH . and MDH by fer-red sem ricytochrome c. (but not with Wurster's blue as electron acceptor) and the same extent of inhibition is seen in the methanol oxidation by whole cells [35,36], this confirms the in vivo nature of the MDH/ferricytochrome c. as­

say system.

Ferricytochrome c. oxidizes reduced MDH with high rates (Chapter VII), so that it seemed unlikely that the disappearance of the methanol-dependent re­ duction of cytochrome c in the anaerobically prepared cell-free extract on

0 admission [24] could be explained from damage to MDH or cytochrome c. To throw more, light on this, it was attempted to isolate the components under anaerobic conditions and to study their 0 sensitivity (Chapter VIII). It appeared that methanol-dependent reduction of ferricytochrome c via MDH re­ quires a mediator. This mediator, probably the "natural" activator for MDH, enhances the substrate oxidation step at pH 7.0 in an assay for MDH with ferricytochrome c as electron acceptor. The factor is present in a complex, which besides cytochrome c , also contains a MDH form with an absorption spectrum different from those already known for the MDH redox forms. Since this form is probably identical to the enzyme as it occurs in the cell, it is indicated as MDH. (MDH in vivo).

TV

The factor can replace artificial activator (NH Cl) in an assay with arti­ ficial electron acceptors, but it is not an NH -salt. The factor can not be replaced by coenzyme A, THF, GSH, Q-9 or a number of divalent metal-ions. In the presence of 0 and cytochrome c (cytochrome c as well as cytochrome c )

d L H

it becomes inactivated.

5.PERSPECTIVES

From the results presented in this thesis, it appears that MDH is an u-nique respiratory-chain linked dehydrogenase. In contrast to other well-known members of this class of enzymes, electrons are not transferred to co­ enzyme Q but to a special cytochrome c. Another striking difference is the requirement of an activator for the substrate oxidation step. The long standing problem of demonstrating the suitability of cytochrome c as an electron acceptor for MDH in vitro seems to be solved now by the discovery

of a factor which may be the natural activator. Structure elucidation of the factor and characterization of the MDH. form are the most urgent topics for research in the near future.

31

-6. REFERENCES

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[2] Attwood, M.M. & Harder, W. (1972) Antonie van Leeuwenhoek 38, 369-378

[3] Dow, C.S. & Whittenbury, R. (1980) in: Contempory Microbial Ecology (Ellwood, D.C., Hedger, J.N., Lothan, M.J., Lynch, J.M. & Slater, J.H., eds.) pp.391-471, Academic Press, London & New York

[4] Hirsch, P. & Conti, S.F. (1964) Arch. Microbiol. 48, 339-357

[5] Meiberg, J.B.M. (1979) Ph.D. Thesis, University of Groningen, The Netherlands

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[7] Anthony, C. in: The Biochemistry of Methylotrophs (1982) Academic Press, London

[8] Stucki, G., Galli, R., Ebersold, H.R. & Leisinger, T. (1981) J. Gen. Microbiol. 130, 366-371

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[10] De Bont, J.A.M., Scholten, A. & Van den Tweel, W.J.J. (1985) Current Microbiol. 12, 267-272

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[17] Salisbury, S.A., Forrest, H.S., Cruse, W.B.T. & Kennard, O. (1979) Nature 80, 843-844

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[19] Dekker, R.H., Duine, J.A., Frank, J.Jzn., Verwiel, P.E.J. & Westerling, J. (1982) Eur. J. Biochem. 125, 69-73

[20] Duine, J.A., Frank, J.Jzn. & Jongejan, J.A. (1987) in: Advances in Enzymology and related Areas of Molecular Biology (Meister, A., ed.), pp.169-212, John Wiley & Sons, New York

[21] Duine, J.A., Frank, J.Jzn. & Westerling, J. (1978) Biochim. Biophys. Acta 524, 277-278

[22] Mincey, T., Bell, J.A., Mildvan, A.S. & Abeles, R.H. (1981) Biochemistry 20, 7502-7509 ■

[23] Duine, J.A., Frank, J.Jzn. & Berkhout, M.P.J. (1984) FEBS Lett. 168,

217-221

[24] Duine, J.A., Frank, J.Jzn. & De Ruiter, L.G. (1979) J. Gen. Microbiol.

115, 523-526

[25] Duine, J.A. & Frank, J.Jzn. (1981) J. Gen. Microbiol. 122, 201-209

[26] Groen, B.W., Frank, J.Jzn. & Duine, J.A. (1984) Biochem. J. 223,

921-924

[27] Groen, B.W., Van Kleef, M.A.G. & Duine, J.A. (1986) Biochem. J. 234,

611-615

[28] Adachi, O., Miyagawa, E., Shinagawa, E., Matsushita, K. & Ameyama, M. (1978) Agric. Biol. Chem. 42, 2331-2340

[29] Adachi, 0.,Tayama, K., Shinagawa, E., Matsushita, K. & Ameyama, M. (1978) Agric. Biol. Chem. 42, 2045-2056

[30] Ameyama, M., Shinagawa, E., Matsushita, K. & Adachi, 0. (1985) Agric. Biol. Chem. 49, 1001-1010

[31] Shimao, M., Yamamoto, H., Ninomiya, K., Kato, N., Adachi, 0., Ameyama, M. & Sakazawa, C. (1984) Agric. Biol. Chem. 48, 2873-2876

[32] Kawai, F., Yamanaka, H., Ameyama, M., Shinagawa, E., Matsushita, K. & Adachi, 0. (1985) Agric. Biol. Chem. 49, 1071-1076

[33] Patel, R.N. & Felix, A. (1976) J. Bacteriol. 128, 413-424

33

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Higgens, I.J. & Quayle, J.R. (1970) Biochem. J. 118, 201-208

Duine, J.A. & Frank, J.Jzn. (1980) Biochem. J. 187, 213-219

Bamforth, C.W., & Quayle, J.R. (1978) Biochem. J. 169, 677-686

Marison, I.W. & Attwood, M.M. (1980) J. Gen. Microbiol. 117, 305-313

Duine, J.A. & Frank, J.Jzn. (1981) in: Microbial Growth on C -compounds Proceedings of the 3th International Symposium. (Dalton, H., ed.) pp.31-41, Heyden, London

De Beer, R., Duine, J.A., Frank, J.Jzn. & Westerling, J. (1983) Eur. J'. Biochem. 130, 105-109

Schaafsma, S.E., Steinberg, H., & De Boer, Th.J. (1966) Rec. Trav. Chim. Pays-Bas 85, 73-74

Schaafsma, S.E., Steinberg, H., & De Boer, Th.J. (1966) Rec. Trav. Chim. Pays-Bas 85, 70-72

Parkes, C. & Abeles, R.H. (1984) Biochemistry 23, 6355-6363

Maclnnes, I., Nonhebel, D.C., Orszulik, S.T. & Suckling, J. (1983) J. Chem. Soc. Perkin Trans.I, 2771-2776

Sherry, B., & Abeles, R.H. (1985) Biochemistry 24, 2594-2605

Anthony, C. (1986) Adv. Microbial. Physio!. 27, 113-203

Anthony, C. (1975) Biochem. J. 146, 289-298

Netrusov, A.I. & Anthony, C. (1979) Biochem. J. 178, 353-360

Netrusov, A.I. (1981) in: Microbial Growth on C -compounds.Proceedings of the 3th International Symposium (Dalton, H., ed.) pp.231-239

Alefounder, P.R. & Ferguson, S.J. (1981) Biochem. Biophys. Res. Commun.

98, 778-784

Jones, C.W., Kingsburry, S.A. & Dawson, M.J. (1982) FEMS Microbiol. Lett. 13, 195-200

Kasprzak, A.A. & Steenkamp, D.J. (1983) J. Bacteriol. 156, 348-353

Beardmore-Gray, M., 0'Keeffe, D.T. & Anthony, C. (1983) J. Gen. Microbiol. 129, 923-933

Ohta, S. & Tobari, J. (1981) J. Biochem. 90, 215-224

[57] Nunn, D.E. & Lidstrom, M.E. (1986) J. Bacteriol. 166, 591-597

[58] 0'Keeffe, D.T. & Anthony, C. (1980) Biochem. J. 190, 481-484

[59] Anthony, C. & Jones, C.W. (1987) in: Microbial Growth on C -compounds . Proceedings of the 5th International Symposium, (Van Verseveld, H.W. & Duine, J.A. eds.) pp. 195-202, Martinus Nijhoff Publishers, Dordrecht [60] Elliott, E.J. & Anthony, C. (1988) J. Gen. Microbiol. 134, 369-377

[61] Beardmore-gray, M., 0'Keeffe, D.T. & Anthony, C. (1982) Biochem. J.

207, 161-165

35

-Chapter II

Monomeric and Dimeric Quinoprotein Alcohol Dehydrogenase from Alcohol-grown Pseudomonas BB1

1.SUMMARY

Pseudomonas BB1, grown on alcohols, contained a quinoprotein alcohol de­

hydrogenase, whose substrate specificity was between those of typical metha­ nol dehydrogenases and ethanol dehydrogenases, and which had a higher turn­ over number and a different amino acid composition. The enzyme occurred in a dimeric as well as in a monomeric form, and the ratio in which the two forms were found depended on the culture conditions. The mechanism of action and the substrate specificity and affinity of the two forms were identical, while the turnover number of the dimer was twice that of the monomer. This

indicates that the catalytic activity of the monomer does not change on di-merization. On inactivating alcohol oxidation of whole cells with cyclopro-panol, monomeric and dimeric enzyme became fully inactivated. It is tenta­ tively concluded that both forms participate in alcohol oxidation in vivo.

2.INTRODUCTION

Gram-negative methylotrophic bacteria oxidize methanol by means of a quinoprotein alcohol dehydrogenase (EC 1.1.99.8), having PQQ as its prosthe­ tic group [1]. Most of these dehydrogenases are dimeric enzymes with a mo­ lecular weight of about 120 000. However, two reports describe the isolation of a monomeric enzyme (molecular weight about 60 000) from methanotrophic bacteria [2,3]. One of these monomeric enzymes was used in mechanistic stud­ ies by Mincey et al. [4] and, surprisingly, the catalytic mechanism proposed

was quite different from the one reported by Duine & Frank [1] for a dimeric enzyme. Although the mechanism proposed by Mincey et al. [4] was refuted for

mono-meric enzyme. Furthermore, a comparative study of monomer and dimer enzyme seemed worthwhile as it could reveal differences which might explain the oc­ currence of the two enzyme forms.

In first attemps to purify the dye-linked alcohol oxidizing activity of

Pseudomonas BB1, two activities appeared to be present. Further

chromato-graphic experiments indicated that one enzyme activity had twice the molec­ ular weight of the other, suggesting that monomeric as well as dimeric en­ zyme was present. Since the occurrence of both enzyme forms in one organism could provide an ideal opportunity for comparative studies, it was decided to purify and characterize the activities.

3.MATERIALS AND METHODS

Cultivation of the organism

Peudomonas BB1 was grown at 30 C on a mineral medium [7] supplemented with

methanol, ethanol or 3-butyn-l-ol (0.2 % v/v). Cells were harvested in the exponential growth phase (0DC,. 1.2-1.4), unless indicated otherwise, washed

DDL)

once with 0.05 M-potassium phosphate, pH 7.0, and stored at -20 C. Continu­ ous growth experiments were done at different dilution rates with the same medium [7].

Purification of the alcohol dehydrogenase activities

Frozen cells cultured on methanol (1 g dry weight) were mixed with 40 ml., 0.05 M-sodium acetate buffer, pH 6.0, and the mixture sonicated (12 times for 15 s, 36 W) with a Branson B12 sonifier. The suspension was centrifuged, (20 min, 48 000 g), yielding the cell extract. The pH of the extract was

lowered to 4.0 with 1 M-HC1 and the precipitate removed by centrifugation. The pH of the supernatant was brought to 6.0 with 1 M-NaOH. After adding

(NH ) SO. to 55 % saturation, the precipitate was removed by centrifugation 4 2 4

37

-the precipitate collected by centrifugation. The precipitate was dissolved in and dialysed against (500 vols, 12 h) 0.02 M-sodium acetate, pH 6.0. The dialysed solution was applied to a CM-Sephadex C-50 cation exchanger (20 x 1.5 cm). The column was washed with portions of 0.02 M-sodium acetate, pH 6.0, containing increasing concentrations of NaCl. The activity eluted at a concentration of 0.15 M-NaCl. Active fractions were pooled, concentrated by pressure filtration and dialysed against 0.1 M-potassium phosphate, pH 7.0. The dialysed solution was chromatographed on a HPLC gel filtration column

(TSK G3000 SW, 7.1 x 600 mm, flow rate 1.0 ml min" ) in 0.1 M-potassium phosphate, pH 7.0. The effluent was monitored with a Hewlett-Packard 1040A photodiode array detector.

Enzyme assay

Alcohol dehydrogenase activity was measured in 1.0 ml assay mixtures, pH 9.0, containing 64 /imol sodium borate, 64 /imol NH Cl, 0.1 /imol Wuster's blue

and 5 /imol ethanol. The reaction was started by adding an appropriate amount

of enzyme and reaction rates were measured at 22 C by following the decrease in absorbance at 600 nm. Wuster's blue was prepared as described by Duine et

3 -1 -1

al. [8], and a molecular absorption coefficient of 9 x 10 -litre.mol . cm

at 600 nm was used in the calculations [9]. Kinetic studies were done by varying the substrate concentrations in the assay system. Apparent kinetic parameters were determined from Lineweaver-Burk double reciprocal plots. For the enzyme purification steps, activities were measured with a Clark-type oxygen electrode. Besides enzyme, the assay mixture (3 ml) contains 150 /imol Tris/HCl buffer, pH 9.0, 0.33 /imol phenazine methosulphate and 45 /imol NH Cl. The reaction was started by adding 15 /imol ethanol.

Inactivation experiments with cyclopropanol

A solution of 50 /il enzyme (approx. 1 nmol) in 0.05 M-NH.C1, pH 9.0, was

in the presence of cyclopropanol. During inactivation, samples were assayed for enzyme activity as described above.

Inactivation experiments with whole cells, harvested in the appropriate growth phase, were done as described by Groeneveld et al [10]. After the

inactivation, the cells were washed twice with 0.05 M-potassium phosphate buffer, pH 7.0, and the enzyme forms isolated according to the isolation procedure described above.

M determinations r

M values of the native enzymes were determined according to Andrews [11], using a Sephadex G-200 column (1 x 53 cm) equilibrated with 0.1 M-sodium phosphate, pH 6.5. Marker proteins were bovine pancreas chymotrypsinogen A

(molecular weight 25 000; Pharmacia), bovine erythrocyte carbonic anhydrase (30 000; Boehringer), ox /J-lactoglobulin (40 000; Boehringer), bovine serum albumine (67 000; Pharmacia), conalbumine (86 000; Serva), rabbit muscle D(-)lactate dehydrogenase (140 000; Boehringer) and yeast alcohol dehydro-genase (150 000; Boehringer).

M values of the subunits were determined on a HPLC gel filtration column r

(Serva Si200 Polyol, 4.1 x 250 mm, flow rate 0.2 ml min ) equilibrated with 0.1 M-sodium phosphate, pH 6.5, containing 0.1 % (w/v) SDS. Proteins (1-2 mg ml ) were denaturated in the presence of 1 % (w/v) SDS and 1 % (v/v)

/J-mer-captoethanol by heating at 100 C for 5 min. After centrifugation (48 000 g,

5 min), 20 /il samples were injected and the effluent monitored at 280 nm. Markers (all from Pharmacia) used in these experiments were rabbit muscle phosphorylase b (moleculair weight 94 000), bovine serum albumine (67 000),

egg white albumine (43 000), bovine erythrocyte carbonic anhydrase (30 000), soybean trypsin inhibitor (20 100) and bovine milk lactalbumin (14 400).

39

-PAGE

This was done on 76 mm 5 % (w/v) polyacrylamide gel slabs, cross-linked with 0.17 % (w/v) bisacrylamide. The gels were electrophoresed in 36mM-Tris/

39 mM-glycine, pH 9.0, or in 36 mM-Tris/21 mM-H PO , pH 6.5, for 1 h at 300V

using a Pharmacia GE-411 electrophoresis apparatus, cooled with tap water. Dehydrogenase activity was detected by soaking the gels in 0.1 M-sodium bo-rate, 0.1 M-NH Cl, 0.05 M-ethanol, pH 9.0, containing an appropriate amount

of Wuster's blue. Protein staining was done with Coomassie brilliant blue R-250.

Analytical methods

Specific absorption coefficients of the enzymes were calculated using the equation derived by Scopes [12] from absorbance measurements at 205 and 280 nm: enzymes were chromatographed on the HPLC column used in the purification and spectra were taken by photodiode array detector at the top of the

elut-Q 1 «

ing enzyme peaks; from this, A " * values were obtained. During the

puri-coU

fication, protein was determined by the Lowry method with bovine serum al-bumine as a standard.

Aromatic amino acids in the preparations were estimated by second-deriv­ ative spectroscopy on a Hewlett-Packard Model 8450 A spectrophotometer ac­ cording to Levine & Frederici [13] after denaturing the proteins with gua-nidine.HCl.

The prosthetic group was analysed as follows. Enzyme solution (50 /xl; approx. 1 nmol) was mixed with 75 /il methanol and adjusted to pH 2.0 with 85 % H,P0.. After centrifugation, the samples were injected onto a C,0

3 4 lo

Radial PAK HPLC column as described by Duine et al. [14], with 85 % H.PO /

H 0/methanol pH 2.0 (0.4:59.6:40, by vol.) as the eluent and a flow rate of

column was calibrated with a known amount of PQQ (molar absorption coeffi-3 - 1 - 1

cient 18.4 x 10 litre.mol . c m ) .

ESR measurements were made as decribed by Duine et al. [8]. Cyclopropanol

was prepared as descibed by Dijkstra et al. [6].

4.RESULTS

Enzyme purification

Table 1. Purification of dye-linked alcohol dehydrogenase from methanol-grown Pseudomonas BB1

Activities were measured with phenazine methosulphate as electron acceptor in a Clark-type oxygen electrode at 30 C. Specific activity is expressed in nmol 0 consumed min .(mg protein)

Purification step Volume Protein Specific Purification Recovery (ml) (mg) activity factor (%) Cell extract Acid treatment (NH ) SO precipitation CM-Sephadex 374 34 25 31 86 136 48 21 220 781 1809 2731 1 3.6 8.2 12.4 100 99 81 54

The results of the purification steps are shown in Table 1. As revealed by the chromatogram (Fig.1), two clearly separated peaks eluted from the gel filtration HPLC column with retention times of 14.5 and 16.6 min. The peaks were collected (fractions I and II, respectively), and both appeared to be

active with a specific activity of 35.3 /unol Wuster's blue reduced min .(mg

dis-I J A ^ , „ = 0-12 II

_JL_

Fig.1. Chromatogram of the HPLC gel filtration step, monitored at 280 nm

Monomeric (II) and dimeric (I) enzymes, had retention times of 16.6 and 14.5 min, respectively.

tinct species which did not interconvert under these chromatographic condi­ tions. Absorption spectra taken upslope, at the top, and downslope of the peaks, were identical. On electrophoresis the fractions showed only one band by activity as well as by protein staining. The band moved to the anode at pH 9.0 (10.5 mm) and to the cathode at pH 6.5 (5.5 mm). Although it is un­ clear why both fractions showed the same.mobility, electrophoresis confirmed that fraction I and II were homogeneous.

Subunit composition

The different retention times of the fractions on HPLC gel filtration sug­ gested a difference in molecular weight. Gel filtration of fraction I.and II on a calibrated Sephadex G-200 column.gave molecular weights of 134 000 ± 7000 and 60 000 ± 3300 respectively. On^the other hand, gel filtration under denaturing conditions gave only one protein peak with a molecular weight of

1-4

1-2

1 0

c

0-8

8

0-6

0-4

0-2

0-0

250 260 270 280 290 300

Wavelength (nm)

Fig.2. UV-absorption spectra of quinoprotein alcohol dehydrogenase in 0.1 M-sodium borate buffer, pH 9.0

The spectra were normalized to a concentration of 0.5 mg protein.ml , Pseudomonas BB1 enzymes (monomeric and dimeric); -.-.-.-., Hyphomicrobium X enzyme; Pseudomonas aeroginosa enzyme.

57 000 + 4700 for both fractions. Although this indicated that the enzyme in fraction I consists of two subunits with the same molecular weight as the enzyme in fraction II, the amino acid composition of the subunits might nev­ ertheless be different. Fig. 2 shows that this is very unlikely since the UV-absorption spectra of the fractions are identical, indicating that at least the aromatic amino acids composition of the fractions was identical (Table 2). Also, the similar electrophoretic mobilities of the two fractions indicates that their overall amino acid compositions are similar. It is con­ cluded, therefore, that the dye-linked alcohol dehydrogenase in this organ­ ism occurs in a dimeric as well as a monomeric enzyme form (fractions I and

0 1 V

43

-as described in MATERIALS AND METHODS, are 2.52 for the dimeric and 2.45 for the monomeric enzyme.

Table 2. Aromatic ami no acid composition of quinoprotein alcohol dehydrogenase

Enzymes (1-2 /JM) were denatured with guanidine.HCl and analysed by deriva­ tive absorption spectroscopy according to Levine & Federici [13]. The Trp and Tyr concentrations were estimated in the 280-300 nm region, while Phe concentration was measured in the 245-265 nm region. In order to compensate for the presence of PQQ, its absorption spectrum was included in the multi-component analysis procedure. In the calculations, molecular weights of 120 000 and 100 000 were used for the enzymes from Hyphomicrobium X and Pseudo-monas aeruginosa, and 114 000 and 57 000 for the dimeric and monomeric en­

zymes from Pseudomonas BB1.

Enzyme source Number of residues per enzyme molecule

Phe Tyr Trp Pseudomonas BB1 (monomeric) Pseudomonas BB1 (dimeric) Hyphomicrobium X Pseudomonas aeruginosa 17 34 41 27 18 36 30 28 18 36 34 33 Prosthetic group

The assay conditions required for activity of the enzyme forms suggested that they belonged to the class of quinoprotein alcohol dehydrogenases. On extraction of the prosthetic group, 2.0 and 0.7 molecules of PQQ per dimer and monomer molecule, were found, respectively. In both enzyme forms as they are isolated, PQQ occurs in its free radical form, as indicated by the (i-dentical) ESR spectra. In accordance with this, reaction with substrate was only observed after the enzymes were converted into their fully oxidized forms by addition of electron acceptor. However, as revealed by absorption