University of Groningen

Physiology and biochemistry of primary alcohol oxidation in the gram-positive bacteria"amycolatopsis methanolica" and "bacillus methanolicus"Hektor, Harm Jan

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Chapter 1

General introduction:Microbial oxidation of primary alcohols

4

General introduction: Microbial oxidation of primary alcohols

1 Introduction

2 Enzymes oxidizing primary alcohols

2.1 Alcohol oxidases (FAD-dependent)

2.2 NAD(P)-independent alcohol dehydrogenases

2.3 NAD(P)-dependent alcohol dehydrogenases

3 Methanol metabolism in bacteria

3.1 Enzymes involved in alcohol oxidation in Gram-negative bacteria

3.2 Enzymes involved in alcohol oxidation in Gram-positive bacteria

3.2.1 Amycolatopsis methanolica

3.2.2 Bacillus methanolicus

3.2.3 Other Gram-positive bacteria

4 NAD-binding

5 Nicotinoproteins

6 Aim and outline of this thesis

General introduction

5

1. IntroductionMethanol is formed in large quantities in nature, mostly from the methyl esters and-ethers that occur in plant components such as pectin and lignin (Dijkhuizen et al.,1992). Methylotrophic micro-organisms able to grow on methanol have beenisolated frequently from soil samples. The techniques employed generally select forthe fastest growing organisms and in most cases have resulted in isolation of purecultures of Gram-negative methylotrophic bacteria (Dijkhuizen et al., 1992). Littleattention has been paid to the large diversity of (relatively slow growing)methylotrophic Gram-positive bacteria (Hazeu et al., 1983; Dijkhuizen et al., 1988;Bastide et al., 1989; Nešvera et al., 1991).

We are interested in the physiology and biochemistry of primary alcoholmetabolism in actinomycetes and Bacilli. A database search showed that few studieshave dealt with the enzymes involved in primary alcohol oxidation in these Gram-positive organisms (Table 1). Previous work in our laboratory has dealt withmethanol metabolism in the thermotolerant bacterium Bacillus methanolicus(Arfman, 1991), providing evidence for the presence of a novel type of methanoloxidizing enzyme (MDH) involved in the metabolism of primary aliphatic alcoholsin general in this organism. Actinomycetes have received most attention because oftheir morphological differentiation and the enormous biochemical diversity of theirsecondary metabolism, amongst others resulting in production of a large variety ofantibiotics (Bibb, 1996). Previous studies in our laboratory have focussed on thephysiology and biochemistry of the main pathways of primary metabolism (Fig. 1)in the nocardioform actinomycete Amycolatopsis methanolica, involved in thebiosynthesis of aromatic amino acids (De Boer, 1990; Euverink, 1995) and inglucose metabolism (Alves, 1997), and on the development of suitable plasmidvectors and transformation systems (Vrijbloed, 1996). The methanol oxidizingenzyme in A. methanolica (Bystrykh et al., 1993a) shares clear similarities with theB. methanolicus MDH enzyme. Both enzymes are characterized in more detail in thepresent study.

Current knowledge about the enzymes involved in primary alcohol oxidation isreviewed in the following paragraphs.

2. Enzymes oxidizing primary alcohols Enzymes oxidizing primary alcohols can be divided in three different groups,depending on the cofactor or coenzyme used:

Chapter 1

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Figure 1. Schematic representation of the pathways used for growth on methanol,glucose and xylose in Amycolatopsis methanolica: assimilatory RuMP cycle (4-9, 14,15), dissimilatory RuMP cycle (4, 5, 10-12), linear dissimilatory pathway (1-3),glycolysis (10, 13-21, and biosynthesis of aromatic amino acids (starting at reaction22) (Alves et al., 1994). The different reactions are catalyzed by the followingenzymes: 1. methanol dehydrogenase; 2. formaldehyde dehydrogenase; 3. formatedehydrogenase; 4. H6P synthase; 5. H6P isomerase; 6. transketolase; 7. transaldolase;8. RuMP epimerase; 9. Ri5P isomerase; 10. G6P isomerase; 11. G6P dehydrogenase;12. 6PGLU dehydrogenase; 13. glucose kinase; 14. phosphofructokinase; 15. FBPaldolase; 16. triosephosphate isomerase; 17. GAP dehydrogenase; 18. 3PG kinase;19. PG mutase; 20. enolase; 21. pyruvate kinase, 22. DAHP synthase. Abbreviations: DAHP, 3-deoxy-D-arabino-heptulosonate-7-phosphate; DHAP,dihydroxyacetonephosphate; E4P, erythrose-4-phosphate; FBP, fructose-1,6-biphosphate; F6P, fructose-6-phosphate; GAP, glyceraldehyde-3-phosphate; G6P,glucose-6-phosphate; H6P, hexulose-6-phosphate; PEP, phosphoenolpyruvate; PYR,pyruvate; 3PG, 3-phosphoglycerate; 6PGLU, 6-phosphogluconate; Ri5P, ribose-5-phosphate; RuMP, ribulose-5-phosphate; S7P, sedoheptulose-7-phosphate; X5P,xylulose-5-phosphate.

General introduction

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2.1. Alcohol oxidases (FAD-dependent)Methylotrophic yeasts (e.g. Hansenula polymorpha) employ an alcohol oxidase(AO; EC 1.1.3.13) that contains FAD as cofactor and is localized in special cell-organelles, the microbody or peroxisome (Harder and Veenhuis, 1989). The AOenzyme catalyzes the oxidation of methanol to formaldehyde and transfers theelectrons derived to oxygen, resulting in hydrogen peroxide formation. This is atoxic compound which subsequently is degraded by catalase. The presence of bothenzymes in peroxisomes may serve to avoid cell damage by the hydrogen peroxideproduced. The subunit size of the usually octameric AO enzymes is 72 to 75 kDaand each subunit contains a noncovalently bound FAD cofactor molecule. AOprotein is synthesized in large amounts during growth under methanol-limitingconditions.

2.2. NAD(P)-independent alcohol dehydrogenasesGram-negative bacteria employ NAD(P)-independent alcohol dehydrogenases(ADH) (Anthony, 1982). Instead of NAD(P), these enzymes use pyrroloquinolinequinone (PQQ), haem and/or F as cofactor. PQQ is typically a cofactor for the420

periplasmic methanol dehydrogenase (MDH) enzymes in methylotrophic Gram-negative bacteria (Duine and Frank, 1980). This is discussed in more detail insection 3.1.

2.3. NAD(P)-dependent alcohol dehydrogenasesThree families of NAD(P)-dependent ADHs (EC 1.1.1.1) have become established(Jörnvall et al., 1987; Reid and Fewson, 1994):- I Long-chain alcohol dehydrogenases- II Short-chain alcohol dehydrogenases-III Iron-dependent alcohol dehydrogenases

Features characteristic of members of Family I are: zinc-dependency, di- ortetrameric quaternary structures and usually a subunit size of 43 kDa. Horse liverADH is a Family I enzyme and has been studied in most detail. Family I ADHsshow no, or relatively low, activities with methanol (Jörnvall et al., 1987; Reid andFewson, 1994).

Members of Family II are metal-independent, possess relatively short primarystructures of on average 240 amino acids, and are usually referred to as “Drosophila-type” enzymes (Krozowski, 1994; Jörnvall et al., 1995). Family II ADHs display abroad substrate specificity and have diverse metabolic roles. There are no reports oftheir involvement in methanol oxidation in methylotrophic bacteria, however.

Chapter 1

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Figure 2. Proposed quaternarystructure of MNO of A.methanolica, based on electronmicroscopy and image processinganalysis (Bystrykh et al., 1993a). A.top view, B. broad side view, C.narrow side view.

Members of Family III initially were referred to as iron-dependent ADHs. Withan increasing number of members of this Family identified, the iron-dependencyappeared not to be a common property, however. Other metal-ions, such as zinc ormagnesium instead of iron, were detected in some of these enzymes. A large numberof ADHs were classified as belonging to Family III on the basis of sequencesimilarity and subunit sizes (352-441 amino acids, on average 391 residues). Arecent database screening for Family III ADHs resulted in identification of a totalof 27 members, 24 of which have been fully sequenced (Chapter 4). Members ofFamily III are found in alcohol-producing and alcohol-consuming micro-organisms,in Gram-negative and Gram-positive bacteria, in anaerobic and aerobic bacteria, inyeasts and amoeba. The various enzymes widely differ in specificity for alcoholsubstrates. Several proteins are part of multifunctional enzymes: aldehyde/alcoholdehydrogenase of Clostridium acetobutylicum (Nair et al., 1994), succinatedegrading enzym-complex in C. kluyveri (Söhling and Gottschalk, 1996),

General introduction

9

alcohol/acetylCoA dehydrogenase of Escherichia coli (Goodlove et al., 1989), anda gene product of Entamoeba histolytica (Yang et al., 1994) of which only the C-terminal half of the deduced sequence shows similarity to ADH. No crystalstructures of Family III ADHs thus far have been reported and only limitedinformation is available about their secondary and tertiary structures. Electronmicroscopic studies and image analysis of five different members revealeddecameric quaternary structures, with two pentameric rings facing each other (Fig.2). This remains a rare feature, however, and appears not to be valid for all FamilyIII ADHs, since independent studies have demonstrated dimeric and tetramericstructures for two other members of Family III: ADH4 of Saccharomyces cerevisiaeand ADHII of Zymomonas mobilis (Williamson and Paquin, 1987; Conway et al.,1987).

Several Family III enzymes from aerobic methylotrophic Gram-positive bacteriaare active with methanol (Table 1) and in vivo catalyze the oxidation of methanolto formaldehyde. These enzymes are reviewed in more detail in section 3.2 and aretopic of further study in this thesis.

3. Methanol metabolism in bacteriaMethylotrophic organisms use one-carbon compounds (e.g. methane, methanol,methylamine) as carbon source for growth. Several specific pathways forassimilation of C substrates have been elucidated in different aerobic methylotrophs1

(Dijkhuizen, 1993) (Fig. 3). Via these pathways carbon-carbon bonds are formed,generating compounds which serve as building blocks for synthesis of cell-material.Autotrophic bacteria synthesize cell material from carbon dioxide, employing theribulose biphosphate (RuBP)- or Calvin cycle. Yeasts follow the xylulosemonophosphate (XuMP) or dihydroxyacetone cycle (not shown). The two other C1

assimilatory pathways identified in bacteria are the serine pathway (assimilatingformaldehyde and CO ) and the ribulose monophosphate (RuMP) cycle2

(assimilating formaldehyde) (Figs. 1, 3). The RuMP cycle is energetically the mostfavourable of these pathways. The Gram-positive bacteria A. methanolica (Hazeuet al., 1983; De Boer et al., 1990a) and B. methanolicus (Dijkhuizen et al., 1988;Arfman et al., 1989, 1992a) both use the RuMP cycle of formaldehyde fixation(fructose bisphosphate aldolase cleavage variant) during growth on methanol,resulting in growth yields of 16-18 g dry weight of cell material/ mole of methanol(Dijkhuizen et al., 1988; De Boer et al., 1990b). These are the highest growth yieldson methanol reported, stimulating considerable interest in possible biotechnologicalapplications (production of single cell protein, enzymes, amino acids, vitamins) withthese bacteria (Schendel et al., 1990; Dijkhuizen, 1993; Euverink, 1995).

Chapter 1

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Table 1. Primary alcohol dehydrogenases in Gram-positive bacteria.

Enzyme Organism Family Gene Metals Subunits Reference

Ethanol:NDMA oxidoreductase Amycolatopsis methanolica I 3 * 39 kDa (Van Ophem et al., 1993)Formaldehyde dehydrogenase Amycolatopsis methanolica I Zn 3 * 40 (Van Ophem et al., 1992b)Alcohol dehydrogenase ADH-T Bacillus stearothermophilus I adhT 4 * 40 (Sakoda and Imanaka, 1992)Alcohol dehydrogenase ADH-hT Bacillus stearothermophilus I adh-hT 4 * 37 (Cannio et al., 1994; Guagliardi et

al., 1996)Glycerol dehydrogenase Bacillus stearothermophilus III gldA ? * 39.5 (Mallinder et al., 1992)Aldehyde/alcohol dehydrogenase Clostridium acetobutylicum III aad ? * 96 (Nair et al., 1994)Alcohol dehydrogenase I Clostridium acetobutylicum III adh1 ? * 43 (Youngleson et al., 1989; Chen, 1995)Butanol dehydrogenase I Clostridium acetobutylicum III bdhB Zn ? * 43 (Welch et al., 1989; Walter et al.,

1992; Petersen et al., 1994;)Butanol dehydrogenase II Clostridium acetobutylicum III bdhA Zn ? * 43 (Welch et al., 1989; Walter et al.,

1992; Petersen et al., 1994)Alcohol dehydrogenase 1 Clostridium beijerinckii B592 III 2 * 42 (Chen, 1995)Alcohol dehydrogenase 2 Clostridium beijerinckii B592 III 42 + 45 (Chen, 1995)Alcohol dehydrogenase 3I Clostridium beijerinckii B592 III 2 * 45 (Chen, 1995)Hydroxybutyrate dehydrogenase Clostridium kluyveri III 4hbd 2 * (42 + 55) (Söhling and Gottschalk, 1996)Alcohol dehydrogenase Mycobacterium bovis BCG I adh Zn 2 * 37.5 (De Bruyn et al., 1981; Stelandre

et al., 1992)Formaldehyde dehydrogenase Rhodococcus erythropolis I Zn 3 * 44 (Eggeling and Sahm, 1985)Alcohol dehydrogenase Rhodococcus rhodochrous 2 * 42 (Ashref and Murrell, 1990)20�-hydroxysteroid dehydrogenase Streptomyces hydrogenans II ? * 26 (Marekov et al., 1990)actIII gene product Streptomyces coelicolor A3(2) II actIII ? * 27 (Hallam et al., 1988; Baker, 1990)? Streptomyces sp. II ? * 28-30 (Freriksen and Heinstra, 1993)Alcohol dehydrogenase Thermoanaerobacter ethanolicus Zn ? * 41.5 (Bryant et al., 1988;

Burdette and Zeikus, 1994)Alcohol dehydrogenase Thermoanaerobacter brockii Zn 4 * 38 (Lamed and Zeikus, 1980, 1981)

enzyme(s) (complexes) active with methanol:Methanol dehydrogenase (n-MDH) Actinomycete strain 381 (Eshraghi et al., 1990)Methanol:NDMA oxidoreductase Amycolatopsis methanolica III mno Mg, Zn 10 * 50 (Bystrykh et al., 1993a, b; Hektor

et al., 1997)methanol dehydrogenase (n-MDH) Amycolatopsis methanolica (Duine et al., 1984a)Alcohol dehydrogenase (MTT-ADH) Amycolatopsis methanolica (Van Ophem et al., 1991)Alcohol dehydrogenase ADH2334 Bacillus stearothermophilus I adh ? * 36 (Dowds et al., 1988; Sheehan et al.,

1988; Robinson et al., 1994)Methanol dehydrogenase Bacillus methanolicus III mdh Mg, Zn 10 * 43 (Arfman et al., 1989)Methanol dehydrogenase Brevibacterium methylicum (Nešvera et al., 1991)Methanol dehydrogenase Corynebacterium. sp XG (Bastide et al., 1989)Methanol:NDMA oxidoreductase Mycobacterium gastri MB19 III Fe, Mg, Zn 10 * 49 (Bystrykh et al., 1993a)Alcohol dehydrogenase (MTT-ADH) Mycobacterium gastri MB19 (Van Ophem et al., 1991)Alcohol dehydrogenase (MTT-ADH) Rhodococcus erythropolis (Van Ophem et al., 1991)Alcohol dehydrogenase (MTT-ADH) Rhodococcus rhodochrous (Van Ophem et al., 1991)ThcE Rhodococcus sp. NI86/21 III thcE ? * 46 (Nagy et al., 1995)

Methylotrophic bacteria generate the metabolic energy required for growth bydissimilating methanol to CO . Two different routes for methanol dissimilation are2

known, the linear pathway and the dissimilatory RuMP cycle (Dijkhuizen, 1993).The linear pathway involves the oxidation of methanol via formaldehyde andformate to CO (Figs. 1, 3). Formaldehyde is a very toxic compound and its2

General introduction

11

Figure 3. Dissimilatory and assimilatory pathways in C -utilizing bacteria (Levering,1

1985).

accumulation above 1 mM is lethal to the cell (Attwood and Quayle, 1984). Theconversion of methanol to formaldehyde therefore requires a sensitive control andalso accurate tuning with the next step, oxidation of formaldehyde to formate. In thedissimilatory RuMP cycle, ribulose 5-phosphate (RuMP) is coupled toformaldehyde, yielding hexulose-6-phosphate (H6P), and in a few steps convertedto glucose 6-phosphate (G6P). The enzymes glucose 6-phosphate dehydrogenaseand 6-phosphogluconate dehydrogenase subsequently produce NAD(P)H andcarbon dioxide, finally also regenerating RuMP (Fig. 1).

3.1. Enzymes involved in alcohol oxidation in Gram-negative bacteriaThe biochemistry of methanol oxidation in Gram-negative methylotrophic bacteriahas been studied in detail. Although a very complex situation exists in theseorganisms, many general characteristics can be discerned. The MDH enzymesemployed are PQQ-dependent, localized in the periplasm and directly connected tothe electron transport chain (Fig. 4). The interactions with different cytochromes areknown in much detail, complete with crystal structures (Anthony, 1992a).

Chapter 1

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Figure 4. Schematic representation of the electron transport system associated withmethanol-oxidation in Gram-negative bacteria. Adapted from Goodwin and Anthony(1995).

MDH is an � � -tetrameric enzyme, with �-subunits of 66 kDa and �-subunits of2 2

8.5 kDa in for instance Methylobacterium extorquens AM1 (Cox et al., 1992). The�-subunit contains one molecule of the PQQ cofactor and one Ca -ion in the active2+

site. Crystal structures show eight radially arranged series of four �-sheets in themain chain of the �-subunit. This propeller like motif encloses PQQ in the activesite and is thought to protect the unstable free radical semiquinone form of PQQ,intermediate of the reaction cycle (Anthony et al., 1994; Goodwin and Anthony,1995). The ß-subunits are wrapped around the �-subunits, rather than formingseparate globular subunits.

Electrons derived from methanol oxidation are transferred to cytochrome cL

(cytochrome c in Paracoccus denitrificans), which is different from other c-type551i

cytochromes, since it is lacking the conserved features (Anthony, 1992b; Dales andAnthony, 1995). Subsequently the electrons are transferred to cytochrome c ,H

cytochrome oxidase and finally to molecular oxygen (Fig. 4). This sequence of stepshas not been confirmed in every organism studied thus far. Many redox mediatorspresent in the periplasmic space are capable of reacting with each other. It istherefore not certain whether the electron transport chain involved is always linear,with more complex networks being possible as well. Mutants lacking specificcytochromes not always provide a clear phenotype, probably because somecytochromes can overtake each others function (Anthony, 1992b). The

General introduction

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concentration of the cytochromes in the periplasmic space is rather high, up to 0.5mM (Anthony, 1986).

The overall methanol oxidation system is very complex, requiring at least 30different gene products. The biosynthesis of PQQ for instance already requires 8gene products. The production of an active methanol dehydrogenase in theperiplasm involves the following steps (Goodwin and Anthony, 1995): synthesis andexport of PQQ, synthesis and export of prepeptides of the �- and �-subunits,processing and folding of these prepeptides, formation of disulphide bridges,insertion of calcium and PQQ, assembly of �-chains around the �-subunits and oftwo ��-units to form the � � -tetramer. Many of the genes involved have been2 2

characterized. Methanol oxidation in Gram-negative bacteria and the nomenclatureof the genes involved have been reviewed by (Lidstrom et al., 1994).

NAD(P)-independent ADHs, not active towards methanol, are somewhat moredivergent. Some ADHs resemble MDH in solubility, subunit composition andhaving cytochrome c as primary electron acceptor (Anthony, 1992a; Schrover et al.,1993; Goodwin and Anthony, 1995). ADH of Comamonas testosteroni containsbesides a PQQ molecule also a haem c molecule. It is therefore called aquinohaemoprotein (Groen et al., 1986; De Jong et al., 1995a, b). Similar solublequinohaemoproteins have been identified in C. acidovorans, Pseudomonas putidaand Rhodopseudomonas acidophila (Toyama, et al., 1995; Yasuda et al., 1996).

Acetic acid bacteria (e.g. Acetobacter aceti, Gluconobacter suboxydans) containa membrane-bound ADH, in quinohaemoprotein complexes, at the periplasmic sideof the cytoplasmic membrane (Matsushita et al., 1992). This enzyme is importantfor the production of acetic acid from ethanol. It consists of three differentcomponents: the haem c and PQQ containing dehydrogenase, a trihaem cytochromec containing subunit, and a third subunit with unidentified prosthetic groups andfunction (Matsushita et al., 1995). The third subunit is missing in the ADH isolatedfrom A. polyoxogenes (Tayama et al., 1989). The various haem moieties areinvolved in the intermolecular electron transfer of ADH to ubiquinone, which isoxidized again by terminal ubiquinol oxidase, cytochrome o or a (Matsushita et al.,1

1992, 1996). Studies of ADH of A. aceti revealed that ADH contains acarboxyterminal extension with the haem c site. Similar to MDH, ADH alsocontains eight series of ß-sheets arranged in a propeller motif (Cozier et al., 1995).

Besides PQQ- and haem-containing ADHs, also NAD(P)-dependent ADHs havebeen identified in Gram-negative bacteria (e.g. Neale et al., 1986; Chen et al., 1989;Hensgens et al., 1993). These NAD(P)-dependent ADHs show no, or relatively low,activity with methanol, however.

Chapter 1

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Figure 5. Hypothetical composition of n-MDH of A. methanolica (Duine et al.,1984a, b).

3.2. Enzymes involved in alcohol oxidation in Gram-positive bacteriaIn contrast to their Gram-negative counterparts, only a limited number of studieshave been done on the biochemistry of primary alcohol oxidation in Gram-positivebacteria. A literature search for general ADHs characterized from Gram-positivebacteria resulted in a list of 30 enzymes (Table 1). Interestingly, all ADHscharacterized in Gram-positive bacteria are NAD(P)-dependent and where studied,soluble, cytoplasmic proteins. The presence of ADHs active with methanol has beenreported in 6 methylotrophic organisms, A. methanolica, B. methanolicus,Mycobacterium gastri, Brevibacterium methylicum, Corynebacterium sp. XG,actinomycete strain 381, and in several non-methylotrophic actinomycetes,oxidizing methanol: Rhodococcus erythropolis, R. rhodochrous, Rhodococcus sp.NI86/21 (Table 1) (Bystrykh et al., 1993b; Chapter 2; Arfman et al., 1989; Nešveraet al., 1991; Bastide et al., 1989; Eshraghi et al., 1990; Van Ophem et al., 1991;Nagy et al., 1995). The methanol-oxidizing enzymes of A. methanolica and B.methanolicus have been studied in most detail and are discussed separately insections 3.2.1 and 3.2.2, respectively. The limited knowledge available about ADHsin other Gram-positive bacteria is briefly summarized in section 3.2.3.

3.2.1. Amycolatopsis methanolicaAmycolatopsis methanolica is a nocardioform actinomycete, showing thecharacteristic formation of a pseudomycelium on solid media, presence of sporesand high GC content of DNA (60-70 mol%) (Kato et al., 1974, 1977; Hazeu et al.,1983; De Boer et al., 1990a). It is the first methylotrophic actinomycetecharacterized, using the RuMP cycle of formaldehyde assimilation (Kato et al.,1977; Hazeu et al., 1983; De Boer et al., 1990b). Over the years it has proven to berather difficult to identify the enzymes involved in methanol oxidation in A.methanolica. Following its isolation from soil of New Guinea, Kato et al. (1975)

General introduction

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Figure 6. Structure ofp-nitroso-N-N'-dimethylaniline.

reported the presence of PMS/DCPIP-dependent MDH activity. Duine et al. (1984a)subsequently provided preliminary evidence for the presence of an NAD-dependent,PQQ-containing MDH (n-MDH), the activity of which could be measured withDCPIP and not with NAD. This activity was detected in methanol-growing cells,producing relatively large amounts of PQQ. It was suggested that n-MDHconstituted a complex of methanol dehydrogenase, NAD-dependent formaldehydedehydrogenase and NADH dehydrogenase activities (Fig. 5) (Duine et al., 1984a).Activities of n-MDH with DCPIP were stimulated by addition of NAD; the complexonly showed activity with methanol, and not with ethanol. The n-MDH complexappeared rather instable, however, with no MDH activity remaining upondissociation in its components. Other quinoproteins have been detected in A.methanolica but thus far no physiological functions could be assigned to any ofthem (Van Ophem and Duine, 1990b).

In the following years it turned out to be rather difficult to reproducibly detect n-MDH activity. The situation considerably improved when van Ophem et al. (1991)reported a novel tetrazolium dye-linked ADH activity in A. methanolica using MTTas an artificial electron acceptor. This enzyme system (MTT-ADH) showed activitywith methanol and various other alcohols. MTT-ADH is considerably more stablethan n-MDH, which allowed its more detailed characterization in subsequentexperiments. Biochemical and mutant evidence showed that MTT-ADH alsoconstitutes a protein complex of three different components, identified asmethanol:NDMA oxidoreductase (MNO, see below) and so-called protein H (withMTT-dependent NADH dehydrogenase activity) and protein L (no separate activityidentified) (Bystrykh et al., 1993b, 1997; Chapter2-4). Following their purification an active MTT-ADH complex could be reconstituted by adding thevarious components together again (Bystrykh et al.,1997).

MNO constitutes the first single protein withmethanol dehydrogenase activity identified in A.methanolica (and in M. gastri) (Bystrykh et al.,1993a, b), using NDMA as artificial electronacceptor (Fig. 6). The molecular characterizationand physiological role of this homo-decamericprotein in A. methanolica is presented in Chapters2-4 (Bystrykh et al., 1993a, b); the data show that the enzyme belongs to Family IIIof NAD(P)-dependent ADHs. It contains a tightly, but non-covalently boundNADP(H) which is redox-active and acts as cofactor. The associated proteins in theMTT-ADH complex most likely function in re-oxidation of the reduced NADPH

Chapter 1

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cofactor. The in vivo electron acceptor for this system in A. methanolica remains tobe identified. The n-MDH and MTT-ADH complexes may share one or morecomponents, although n-MDH is highly specific for methanol and MTT-ADHshows a much broader substrate specificity (various primary and secondaryalcohols).

A separate ethanol:NDMA oxidoreductase (ENO) activity has been identified inA. methanolica (Van Ophem et al., 1993). This trimeric enzyme, with subunits of39 kDa, contains a tightly bound NAD as cofactor and belongs to Family I. Theenzyme is most active in methanol-grown cells, although it is not active withmethanol. The in vivo role of ENO is unclear. Mutants lacking MNO, but stillpossessing ENO, were unable to grow on methanol, ethanol, propanol or butanol(Chapter 3).

Five different formaldehyde oxidizing enzymes have been identified in A.methanolica (Van Ophem, 1993):

1. NAD-dependent aldehyde dehydrogenase (NA-AlDH)(Van Ophem and Duine, 1990a)

HCHO + NAD â HCOOH + NADH + H+ +

2. NAD- and factor-dependent formaldehyde dehydrogenase (FD-FAlDH)(Van Ophem and Duine, 1990a; Van Ophem et al., 1992b)

HCHO + NAD â HCOOH + NADH + H+ +

3. Formate ester dehydrogenase (FEDH) (Van Ophem et al., 1992a)ROCHO + Wb â ROCOOH + Wb ox red

(R = alkyl group; Wb = Wurster’s blue)

4. Dye-linked aldehyde dehydrogenase (DL-AlDH)(Van Ophem et al., 1992a; Kim et al., 1996)

HCHO + DCPIP â HCOOH + DCPIPox red

5. Formaldehyde dismutase (MNO) (Bystrykh et al., 1993b; Chapters 2-4)HCHO + HCHO â CH OH + HCOOH3

Each of these enzymes has been purified and characterized. Purified MNO showshigh formaldehyde dismutase activities. The in vivo significance of this activityremains doubtful. Formaldehyde dismutase is known to increase the resistance ofP. putida to formaldehyde (Kato et al., 1983; Yanase et al., 1995) but A.methanolica is very sensitive to the toxic effects of formaldehyde (De Boer et al.,1990b). Mutants lacking MNO are still capable of growing on formaldehyde as solecarbon- and energy source (in formaldehyde-limited chemostat cultures) (Chapters

General introduction

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3, 4). The strongest candidate for an in vivo role in formaldehyde degradation wasFD-FAlDH. This enzyme is only found in methanol-grown cells and is specific forformaldehyde. The other formaldehyde-oxidizing enzymes also are active withhigher aldehydes (Van Ophem et al., 1992b). However, a methanol-negative mutantof A. methanolica which lacked not only MNO but also FD-FAlDH and NA-AlDHcould still grow on formaldehyde, displaying an increased level of DL-AlDH(Chapter 3). FD-FAlDH is a trimeric enzyme of 117 kDa, containing Zn , and also2+

active with ethanol but not with methanol. A similar activity has been found in R.erythropolis (Eggeling and Sahm, 1985); these enzymes are comparable to theNAD/glutathione-dependent formaldehyde dehydrogenase of eukaryotes and Gram-negative bacteria (Van Ophem and Duine, 1994). The N-terminal sequence of FD-FAlDH shows about 30 % similarity with Family I ADHs (Jörnvall et al., 1987;Julià et al., 1988; Van Ophem et al., 1992b). The identity of the heat-stable factorremains unknown, but can be mimicked by high concentrations of methanol (VanOphem et al., 1992b). This led to the hypothesis that the true substrate for FD-FAlDH is a hemiacetal of methanol and formaldehyde (CH -O-CH OH) and that3 2

methylformate is the product, possibly an intermediate in an alternativeformaldehyde oxidation pathway (Van Ophem et al., 1992a; Sakai et al., 1995).Methylformate or factor-formate would be the substrate for FEDH. Althoughmethanol is not a substrate for FD-FAlDH, the enzyme is induced by growth onmethanol. In contrast, ethanol did not induce FD-FAlDH, but NA-AlDH (VanOphem and Duine, 1990a). This is a homotetramer of 200 kDa, with a broadsubstrate specificity, but not active with formaldehyde, although it is also inducedduring growth on methanol (Van Ophem and Duine, 1990a).

FEDH and DL-AlDH are dye-linked enzymes; their in vitro activity is assayedwith artificial electron acceptors since the biological acceptors are unknown. FEDHis a molybdoprotein, containing 1 Mo, 4 Fe, 3 or 4 S, and 1 FAD per enzymemolecule of 186 kDa (Van Ophem et al., 1992a; Kim et al., 1996). The enzyme isinduced when growing on primary alcohols, with methanol and 1-hexanol as thebest inducing substrates. The enzyme is active with aldehydes and formate esters(esters of formate and methanol or ethanol), but not with alcohols or formateseparately.

DL-AlDH is also a molybdoprotein, containing FAD, Fe and S. Just like FEDHand molybdoproteins in general, DL-AlDH consists of three different subunits, witha total mass of 160 kDa (87, 35 and 17 kDa) (Kim et al., 1996). In contrast toFEDH, with � � � subunit composition, DL-AlDH has an unique � � � structure.2

FEDH and DL-AlDH are two different enzymes, although their induction patternand substrate specificity suggest a similar physiological role.

Chapter 1

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Except for formaldehyde dismutase (MNO), none of the above formaldehyde-oxidizing enzymes have been found associated with the MTT-ADH complex.

3.2.2. Bacillus methanolicusBacillus methanolicus is a thermotolerant methylotrophic Gram-positive bacterium(Dijkhuizen et al., 1988; Arfman et al., 1992a).The organism was isolated for itscapability to grow on methanol at elevated temperatures. B. methanolicus can growon temperatures up to 60°C, with an optimum between 50 and 55°C. B.methanolicus also possesses the RuMP pathway for formaldehyde fixation(Dijkhuizen et al., 1988). Electron microscopic studies and image processingrevealed that B. methanolicus also possesses a decameric alcohol-oxidizing enzyme(Vonck et al., 1991). In contrast to MNO of A. methanolica, the methanoldehydrogenase of B. methanolicus (MDH) is active with NAD as coenzyme(Arfman et al., 1989). Cells grown under methanol-limiting conditions accumulateMDH up to 22 % of total soluble protein (Arfman et al., 1989), probably toovercome the poor affinity of MDH for methanol. MDH contains Mg and Zn2+ 2+

instead of iron (Vonck et al., 1991). MDH contains a tightly, but non-covalentlybound NAD; further studies showed that this NAD(H) is redox-active and functionsas a cofactor (Arfman et al., 1997; Chapter 5). The mdh gene has beencharacterized, resulting in classification of MDH as a Family III enzyme (De Vrieset al., 1992).

B. methanolicus possesses a special activator protein, consisting of two subunitsof 27 kDa, which stimulates the relatively low activity of MDH towards methanol(Arfman et al., 1991). This stimulation is Mg -dependent and results in a 40-fold2+

increase in MDH turnover rate. In Chapters 5 and 6 we provide evidence that theactivator protein stimulates re-oxidation of the MDH cofactor, being the rate-controlling step. The gene encoding this protein has been characterized(Kloosterman et al., 1997); the data show that the activator protein is a member ofthe MutT family (Koonin, 1993). The E. coli MutT protein is a Mg -dependent2+

triphosphatase, hydrolyzing 8-oxo-dGTP as preferred substrate. The nucleotide 8-oxo-dGTP can pair with cytosine as well as adenine, causing errors in DNAreplication. MutT degrades 8-oxo-dGTP into 8-oxo-dGMP and pyrophosphate,thereby preventing these mutations to occur (Bullions et al., 1994). The mechanismof the B. methanolicus activator protein remains to be studied in more detail. In vivothis protein may have an important physiological role, contributing to the control ofMDH activity. MDH is synthesized in abundant amounts, making its control adelicate problem since accumulation of formaldehyde, the product of the MDHreaction, is lethal to the cell (Arfman et al., 1992b).

General introduction

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3.2.3. Other Gram-positive bacteriaADHs have been identified and characterized in a few other Gram-positive bacteria.Three different ADHs have been isolated from three different Bacillusstearothermophilus strains. ADH2334 of strain DSM 2334, an obligately aerobicorganism, possesses activity with methanol (K 20 mM). The enzyme is substratem

inhibited and is thought to function in alcohol oxidation in vivo (Dowds et al., 1988;Sheehan et al., 1988; Robinson et al., 1994). This is one of the very few methanol-oxidizing ADHs of Family I (Sheehan et al., 1988). ADH-T of strain NCA 1503,a facultatively anaerobic, ethanologenic strain (Sakoda and Imanaka, 1992), is athermostable enzyme, inactive with methanol, insensitive to substrate inhibition, andinvolved in ethanol production. The even more heat stable ADH-hT, isolated ofstrain NCIMB 12403, is also inactive with methanol and involved in ethanolproduction (Guagliardi et al., 1996). Sequence data show an high degree of identity,but biochemical and immunological data clearly suggest that these three enzymesare different (Sheehan et al., 1988; Sakoda and Imanaka, 1992; Cannio et al., 1994;Robinson et al., 1994; Guagliardi et al., 1996). All three proteins belong to FamilyI and have subunit sizes of approximately 36 kDa. A single member of Family IIIADHs thus far has been identified in a Bacillus species other than B. methanolicus.Interestingly, this is a glycerol dehydrogenase, encoded by the gldA gene of B.stearothermophilus var. non-diastaticus (Mallinder et al., 1992).

Corynebacterium sp. XG is a facultative methylotroph, which assimilatesmethanol carbon via the serine pathway (Bastide et al., 1989). Methanoldehydrogenase activity could be detected with phenazine methosulphate as artificialelectron acceptor. Brevibacterium methylicum assimilates methanol carbon via theRuMP cycle. This organism possesses NAD-dependent MDH activity (Nešvera etal., 1991). The methylotrophic actinomycete strain 381 was reported to possess ann-MDH activity (Eshraghi et al., 1990), comparable to A. methanolica (Duine et al.,1984a). The further characterization of these methanol oxidizing enzymes has notbeen reported.

Most ADHs have been characterized from various Clostridium species. Mucheffort has been devoted to the characterization of these enzymes, mainly because oftheir involvement in production of the commercially interesting butanol and 2-propanol. C. acetobutylicum contains several different ADHs. First of all there aretwo butanol dehydrogenases (BDHI and BDHII). These isoenzymes prefer NADHabove NADPH as coenzyme (Walter et al., 1992; Petersen et al., 1994), althoughChen (1995) reported that this preference is pH-dependent. The gene adh1 codes fora different NADPH-dependent ADH involved in the production of butanol andethanol (Youngleson et al., 1989), while aad codes for a bifunctional enzyme (Nairet al., 1994). The N-terminal part of the protein shows similarity with aldehyde

Chapter 1

20

dehydrogenases, while the C-terminal half of the protein encodes an alcoholdehydrogenase (Nair et al., 1994).

C. beijerinckii contains at least three NADPH-dependent ADH isoenzymes.ADH1 is a homodimer of 42 kDa �-subunits, ADH2 is a heterodimer of a single �-subunit and a 45 kDa �-subunit, and ADH3 is a homodimer of �-subunits (Chen,1995). The number of strains studied and the rather different methods used forprotein characterization make it difficult to establish clear resemblances anddifferences between the ADHs of Clostridia. Data presented thus far indicate thatall primary ADHs of Clostridium species belong to Family III (Table 1). This alsoincludes the butyraldehyde dehydrogenase involved in succinate degradation by C.kluyveri, which is part of a bifunctional protein as also reported for thealcohol/aldehyde dehydrogenase of C. acetobutylicum(Söhling and Gottschalk,1996; Nair et al., 1994).

Thermoanaerobacter ethanolicus contains a primary ADH with a temperatureoptimum of 85°C (Bryant et al., 1988; Burdette and Zeikus, 1994). The enzyme isa homotetramer of 41.5 kDa subunits and contains zinc. It has been hypothesizedthat a secondary ADH, also active with ethanol and propanol, is responsible formost of the ethanol production, while the primary ADH is involved in the ethanolconsumption (Bryant et al., 1988). The catalytic efficiency of the primary ADH forNADH oxidation is eight times higher than for NADPH oxidation and the reductionof NADP is 30 times more efficient than NAD reduction. Correspondingefficiencies for acetaldehyde reduction and ethanol oxidation suggest that theprimary ADH can control the NADH and NADPH pools by cycling betweenacetaldehyde and ethanol (Lovitt et al., 1988; Burdette and Zeikus, 1994). Acomparable situation has been found in Thermoanaerobacter brockii (Lamed andZeikus, 1980, 1981). No sequence information is available for these enzymes; inview of their molecular weights and presence of zinc they are either Family I orFamily III ADHs.

Several Family II ADHs have been identified in the genus Streptomyces. One ofthese, 20�-hydroxysteroid dehydrogenase, has been characterized from S.hydrogenans (Marekov et al., 1990). A second one, the actIII gene product, hasbeen found in S. coelicolor A3(2) and is involved in biosynthesis of the polyketideantibiotic actinorhodin (Hallam et al., 1988; Baker, 1990). Family II ADHs may bemore widespread in Streptomyces. Antibodies raised against Drosophila ADH(Family II) showed no cross-reactivity with the 20�-hydroxysteroid dehydrogenasebut recognized a 28-30 kDa protein in several Streptomyces species, e.g. S. lividans(Freriksen and Heinstra, 1993). This protein became more abundant in a latergrowth phase, indicating its possible involvement in the production of secondary

General introduction

21

Figure 7. Mode of action of alcohol dehydrogenase. X indicates electron acceptor.

metabolites or in morphological differentiation. Since the actIII-product wasmissing in S. lividans its 28-30 kDa protein should be another Family II ADH.

ADH of Mycobacterium tuberculosis var. bovis (BCG) has been partially purifiedand characterized as a dimer with subunits of 37.5 kDa, containing zinc (De Bruynet al., 1981). Highest affinity was observed with butyraldehyde (125 µM); theaffinity for butanol was 220 mM. Based on the deduced amino acid sequence of adhthe enzyme was classified as a Family I ADH (Stelandre et al., 1992).

Various ADHs have been characterized from Rhodococcus species. R.rhodochrous possesses a secondary ADH, which shows poor activity with primaryalcohols, but not with methanol (Ashref and Murrell, 1990). This NAD-dependentenzyme is a dimer with subunits of 42 kDa. R. erythropolis and A. methanolicacontain similar factor-dependent NAD-dependent formaldehyde dehydrogenases(FD-FAlDH) (Family I) (Eggeling and Sahm, 1985; Van Ophem et al., 1992b).

The ThcE enzyme of Rhodococcus sp. NI86/21 is very similar to MNO of A.methanolica and M. gastri (Bystrykh et al., 1993a, b; Nagy et al., 1995; Chapters2-4). MNO has a key function in methanol oxidation in the latter twomethylotrophic bacteria, but Rhodococcus sp. NI86/21 is unable to grow onmethanol. The ThcE protein is induced during growth with thiocarbamate and isthought to be involved in the biodegradation of this and other herbicides (Nagy etal., 1995).

MTT-dependent alcohol dehydrogenase (MTT-ADH) activities have beenreported not only for A. methanolica, but also for M. gastri, R. erythropolis and R.rhodochrous (Van Ophem et al., 1991). MTT-ADH of A. methanolica consists ofa complex of at least 3 proteins including MNO (see above). MNO has also beenidentified in M. gastri and Rhodococcus species, but it is not clear whether MTT-ADH activity can be assigned to a comparable complex in these organisms.

4. NAD-bindingAlcohol dehydrogenase enzymes oxidize an alcohol group of their substrates bytransfer of a hydride-ion from the carbon atom that binds the hydroxyl group to theoxidized form of their coenzymes or cofactors. In addition, a proton is removedfrom the alcohol hydroxyl group (Fig. 7). During dehydrogenation this hydride-ion

Chapter 1

22

Figure 8. Schematic drawing of the ���-fold (Rossmann-fold) and the binding toNAD. The amino acid sequence indicated (in single letter code) is of lactatedehydrogenase (Eventoff et al., 1977; Taylor, 1977). The fingerprint consists of threeGly-residues (double circle), one basic- or hydrophilic residue (�), six small- andhydrophobic residues (*), and one acidic residue (�) which forms hydrogen bondswith the 2' OH group of the ribose-moiety of NAD (Wierenga et al., 1986).

and proton are directly transferred from substrate to coenzyme/cofactor. Theseenzymes, therefore, not only must recognize and bind substrate and for instanceNAD(P), but also must position them in the active site, sufficiently close and in thecorrect orientation to allow direct hydrogen transfer to take place. The dinucleotide-binding domains of various dehydrogenases have very similar three-dimensionalstructures, although most of the amino acid side-chains that interact with NAD(P)can vary (Lesk, 1995). The majority of these proteins contains a Rossmann- or ���-fold (Fig. 8), which is involved in binding of the ADP moiety present in NAD(P)and FAD (Fig. 9) (Rossmann et al., 1974). This structural fold of about 30-35residues can be obtained from many different amino acid sequences. Consequently,there is not a single consensus motif for every NAD- or FAD-binding domain and

General introduction

23

Figure 9. Structures of NAD and FAD, both possessing the ADP-moiety.

the residues involved may differ widely in different proteins of the same organism,and also between similar proteins of different organisms (for a review Brandon andTooze, 1991). Nevertheless, there are strong stereochemical constraints at specificpositions in the polypeptide chain of the Rossmann-fold that must be respected topreserve its structure and function. These invariant key residues provide afingerprint to predict dinucleotide-binding regions in proteins of known amino acidsequence but with unknown three-dimensional structure (Wierenga et al., 1986).Most recognizable is the Gly rich GXGXXG motif (X, variable amino acid). Ananalysis of 133 sequences of FAD- or NAD-dependent proteins (Nishiya andImanaka, 1996) confirmed the presence of the conserved GXGXXG motif. Inaddition, in 56 sequences (42 %) the second position was occupied by a Gly or Alaresidue, resulting in G(GA)GXXG. Large, acidic, basic or aromatic amino acids didnot appear so often at this second position. The GXGXXG motif, together with sixconserved hydrophobic amino acids, enables formation of a ��� fold, alsopositioning an Asp or Glu residue that forms a hydrogen bond with the 2'-OH of theADP-moiety of NAD and FAD (Figs. 8, 9). The 2'-OH position in NADP(H) isoccupied by a phosphate group; in an NADP-binding domain the Asp or Glu residuehas usually been replaced by a residue with a smaller side-chain, such as Gly(Brandon and Tooze, 1991). Characteristic for an NADP(H)-binding domain also

Chapter 1

24

is that the third Gly of the GXGXXG motif generally has become replaced by Ala(Scrutton et al., 1990). These are not the only two residues determining NAD(P)(H)coenzyme specificity, however. Seven point mutations (A179G, A183G, V197E,K199F, R198M, H200D, R204L) were required to achieve a clear shift in coenzymespecificity of NADP-dependent glutathione reductase of E. coli towards NAD(Scrutton et al., 1990; Mittl et al., 1993, 1994). Single mutations did cause a shiftin coenzyme specificity of this protein but these mutants were catalytically lessefficient than the wild type enzyme. Not only amino acid side-chains but also mainchain NH-groups may form hydrogen-bonds with the coenzyme, which diminishesthe effects of point mutations (Baker et al., 1992).

Although the GXGXXG motif is well conserved, some variations do occur (Lesk,1995). In malate dehydrogenase of E. coli an extra Ala is inserted after the first Gly(GAXGXXG), causing a local deformation of the loop between the first ß-sheet andthe �-helix. Also 20�-hydroxysteroid dehydrogenase of S. hydrogenans contains aninsertion in the loop, yielding the sequence pattern which is conserved in ADHs ofFamily II: GXXXG(A)XG (Ghosh et al., 1991; Lesk, 1995). In NADP-dependent6-phophogluconate dehydrogenase of sheep and dihydropteridine reductase of ratthe position of the second Gly is occupied by an Ala residue, which causes adifferent interaction between enzyme and coenzyme (Shahbaz et al., 1987; Lesk,1995).

Further variations in the Gly rich motif for NADP-binding (GXGXGXXPF) andfor NAD-binding (GGXGXFP), still allowing formation of ���-folds, were foundin a family of NAD(P)-binding flavoproteins (Karplus et al., 1991; Bredt et al.,1991; Segal et al., 1992). Although also the FAD-binding domains of these proteinsdid show some sequence similarity, only a Ser residue was fully conserved. Thisgroup includes the human proteins NADPH-cytochrome b and NADH-245

cytochrome b reductase, the rat proteins NADPH-cytochrome P-450 reductase and5

NADPH-nitric oxide synthase (Bredt et al., 1991), NADH-nitrate reductase oftomato, ferredoxin-NADP reductase (FNR) of spinach, NADPH-sulphite reductaseof Salmonella typhimurium, and NADPH-cytochrome P-450 reductase fromBacillus megaterium (Segal et al., 1992). The atomic structure of FNR demonstratedan antiparallel ß-barrel core and a single �-helix as unusual binding domain for thepyrophosphate of FAD (Karplus et al., 1991). The actual interactions between FADand the protein are with Arg, Ser, Tyr residues, and the peptide amides at theaminoterminal end of the �-helix. The presence of a Gly residue again allows aclose approach of the helix to the pyrophosphate group. A total of 6 peptidesegments involved in FAD and NADP-binding could be identified; these areconserved in enzymes of the FNR-family. Also the spacing between these segments

General introduction

25

is the same, this conservation is significant, although the separate segments are tooshort to indicate individual similarities (Karplus et al., 1991).

The NAD(P)-binding domains in ADHs of Families I and II generally can beidentified straightforwardly, when searching for the GXGXXG fingerprint. Only afew members of Family III ADHs possess this classical dinucleotide-bindingdomain (De Vries et al., 1992; Chapter 4). Chapter 6 describes the characteristicsof site-directed mutants of MDH of B. methanolicus, allowing identification of anew NAD(P)(H)-binding domain in members of Family III ADHs.

5. NicotinoproteinsNAD(P) functions as a coenzyme for a large variety of dehydrogenase enzymes,receiving or donating electrons depending on the specific reaction catalyzed and thereaction conditions. The cytosolic NAD(P)(H) can be oxidized or reduced elsewherein the cell, e.g. by NAD(P)H dehydrogenase in the cytoplasmic membrane. In recentyears a limited number of so-called nicotinoproteins, containing tightly butnoncovalently bound NAD(P) (Van Ophem, 1993) have become recognized.Analogous to for instance FAD in flavoproteins and PQQ in quinoproteins,NAD(P)(H) may also be acting as a cofactor in nicotinoproteins and remains boundto the enzyme during catalysis. An external electron donor or acceptor subsequentlymay reduce or oxidize the cofactor in situ. Zymomonas mobilis for instance couplesthe oxidation of glucose to the reduction of fructose using a periplasmic glucose-fructose oxidoreductase which contains tightly bound NADP(H) as cofactor(Zachariou and Scopes, 1986; Kanagasundaram and Scopes, 1992; Loos et al.,1993). Examples of NAD(P)-containing nicotinoproteins are lactate:oxaloacetateoxidoreductase of Veillonella alcalescens (Allen, 1966, 1982), UDP-galactose 4-epimerase from E. coli (Bauer et al., 1992) and formaldehyde dismutase of P. putidaF61 (Kato et al., 1986). The latter enzyme serves to strongly increase formaldehyderesistancy of the cells, coupling oxidation of one formaldehyde molecule to formatewith reduction of a second formaldehyde molecule to methanol (Kato et al., 1986).

Studies of methanol-utilizing Gram-positive bacteria have resulted inidentification of several nicotinoproteins in recent years. MNO (Bystrykh et al.,1993a) and ENO (Van Ophem et al., 1993) of A. methanolica, and MNO of M.gastri MB19 (Bystrykh et al., 1993a), are nicotinoproteins. Both MNO (Family III)and ENO (Family I) catalyze the NDMA-linked oxidation of alcohols, similar to P.putida formaldehyde dismutase. MNO (but not ENO (Van Ophem et al., 1993))dismutates formaldehyde (Bystrykh et al., 1993b; Chapter 2) but its physiologicalrole appears to be in oxidation of primary alcohols (Chapters 3, 4). The in vivoelectron acceptor for MNO remains to be identified. Also MDH of B. methanolicus

Chapter 1

26

is a nicotinoprotein (Arfman, 1991; Arfman et al., 1997; Chapter 5). MNO in A.methanolica is part of a three component protein complex with MTT-ADH activity(Bystrykh et al., 1997). Re-oxidation of the reduced NAD(P)H cofactors in MDHand MNO appears to involve interaction with other cytoplasmic proteins,phenomena that are subject of further study in this thesis.

6. Aim and outline of this thesisAim of this thesis was to investigate the physiology and biochemistry of oxidationof methanol (and other primary alcohols) in Gram-positive bacteria. Most of thecurrent knowledge of methanol oxidation is based on studies with Gram-negativemethylotrophs and the number of detailed studies of their Gram-positivecounterparts is limited. The methanol-oxidizing systems in Gram-negative bacteriaare located in the periplasm and use PQQ as cofactor. This prompted questionsabout the nature and location of these systems in Gram-positive organisms whichlack a clear periplasmic space and generally do not possess PQQ. The studiesdescribed in this thesis focussed on two methanol-utilizing bacteria, theactinomycete A. methanolica and the thermotolerant bacterium B. methanolicus.Previous studies suggested that these bacteria employ novel ADH protein complexesin the metabolism of lower primary aliphatic alcohols (Duine et al., 1984a, b; VanOphem et al., 1991; Arfman et al., 1991). Interestingly, several reports also provideevidence for the presence of PQQ in A. methanolica; its physiological role hasremained unclear, however (Hazeu et al., 1983; Duine et al., 1984a; Van Ophemand Duine, 1990b). Preliminary evidence also indicated that these novel enzymesoccur more widespread in actinomycetes. Various approaches were followed,involving application of (at random and site-directed) mutagenesis, biochemicaltechniques (protein purification and characterization) and molecular techniques(gene cloning, site-directed mutagenesis), to characterize these enzyme systems inmore detail.

Chapter 1 reviews current knowledge of microbial primary alcohol oxidation,with emphasis on Gram-positive bacteria. The purification and characterization ofan A. methanolica enzyme with high formaldehyde dismutase activity, but alsooxidizing methanol and other primary alcohols, is described in Chapter 2 (Bystrykhet al., 1993a, b). This methanol:NDMA oxidoreductase (MNO) shows no activitywith coenzyme NAD(P) but otherwise shares similarities with the previouslydescribed NAD-dependent methanol dehydrogenase (MDH) of B. methanolicus(Arfman et al., 1989; Vonck et al., 1991). Both enzymes are for instance decamericnicotinoproteins containing NAD(P)(H) cofactors (Bystrykh et al., 1993a). MNOappears to be part of a protein complex with proteins H and L, showing MTT-

General introduction

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dependent ADH activity (Bystrykh et al., 1997). The characterization of A.methanolica mutants unable to grow on methanol provided clear evidence thatMNO and protein H are essential for the metabolism of primary alcohols in general(Chapter 3). Cloning and characterization of the MNO-encoding gene allowedconstruction of the A. methanolica derivative strain MDM2 with a disrupted mnogene (single cross-over). Characterization of this mutant strain confirmed that lossof MNO resulted in complete failure to grow on primary alcohols; growth onformaldehyde remained possible, however. Sequence alignments showed that MNO(and MDH) are members of the steadily growing Family III of NAD(P)-dependentADHs (Chapter 4).

Further studies concentrated on the functions and mode of binding of the NAD(P)cofactors present in each subunit of MDH of B. methanolicus and MNO of A.methanolica (Bystrykh et al., 1993a; Arfman et al., 1997; Chapter 5). Biochemicalstudies of the reaction cycle of the B. methanolicus MDH demonstrated that itstightly, but non-covalently, bound NAD is redox-active and remains bound duringcatalysis. An exogenous, coenzyme NAD molecule, is able to re-oxidize the reducedNADH cofactor, resulting in relatively low NAD-MDH activities. This step isfacilitated by a B. methanolicus activator protein of 50 kDa (Arfman et al., 1991)which strongly stimulates MDH turnover in the presence of NAD and Mg -ions.2+

Alignments of the full length sequences of the 24 Family III ADHs currentlyknown initially failed to identify the classical dinucleotide-binding fold forNAD(P)(H). Three unique, conserved sequence motifs, were detected in theseproteins, however. Following site-directed mutagenesis of MDH (Chapter 6),evidence was obtained for the involvement of several amino acids in one of thesemotifs in NAD cofactor and coenzyme-binding. This conserved motif thus may bepart of a new NAD(P)-binding fold in Family III ADHs.

ReferencesReferences are listed on pages 127 - 136.