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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Download date: 29-09-2021 Chapter 1

General introduction: Microbial oxidation of primary alcohols

3 General introduction: Microbial oxidation of primary alcohols

1 Introduction 2 oxidizing primary alcohols 2.1 Alcohol oxidases (FAD-dependent) 2.2 NAD(P)-independent alcohol dehydrogenases 2.3 NAD(P)-dependent alcohol dehydrogenases 3 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

4 General introduction 1. Introduction Methanol 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 been isolated frequently from soil samples. The techniques employed generally select for the fastest growing organisms and in most cases have resulted in isolation of pure cultures of Gram-negative methylotrophic bacteria (Dijkhuizen et al., 1992). Little attention 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 alcohol metabolism in actinomycetes and Bacilli. A database search showed that few studies have dealt with the enzymes involved in primary alcohol oxidation in these Gram- positive organisms (Table 1). Previous work in our laboratory has dealt with methanol metabolism in the thermotolerant bacterium Bacillus methanolicus (Arfman, 1991), providing evidence for the presence of a novel type of methanol oxidizing (MDH) involved in the metabolism of primary aliphatic alcohols in general in this organism. Actinomycetes have received most attention because of their morphological differentiation and the enormous biochemical diversity of their secondary metabolism, amongst others resulting in production of a large variety of antibiotics (Bibb, 1996). Previous studies in our laboratory have focussed on the physiology and biochemistry of the main pathways of primary metabolism (Fig. 1) in the nocardioform actinomycete Amycolatopsis methanolica, involved in the biosynthesis of aromatic amino acids (De Boer, 1990; Euverink, 1995) and in glucose metabolism (Alves, 1997), and on the development of suitable plasmid vectors and transformation systems (Vrijbloed, 1996). The methanol oxidizing enzyme in A. methanolica (Bystrykh et al., 1993a) shares clear similarities with the B. methanolicus MDH enzyme. Both enzymes are characterized in more detail in the present study. Current knowledge about the enzymes involved in primary alcohol oxidation is reviewed in the following paragraphs.

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

5 Chapter 1

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 reaction 22) (Alves et al., 1994). The different reactions are catalyzed by the following enzymes: 1. methanol dehydrogenase; 2. dehydrogenase; 3. formate dehydrogenase; 4. H6P synthase; 5. H6P ; 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. FBP aldolase; 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.

6 General introduction

2.1. Alcohol oxidases (FAD-dependent) Methylotrophic yeasts (e.g. Hansenula polymorpha) employ an (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 AO enzyme catalyzes the oxidation of methanol to formaldehyde and transfers the electrons derived to , resulting in hydrogen peroxide formation. This is a toxic compound which subsequently is degraded by catalase. The presence of both enzymes in peroxisomes may serve to avoid cell damage by the hydrogen peroxide produced. The subunit size of the usually octameric AO enzymes is 72 to 75 kDa and each subunit contains a noncovalently bound FAD cofactor molecule. AO protein is synthesized in large amounts during growth under methanol-limiting conditions.

2.2. NAD(P)-independent alcohol dehydrogenases Gram-negative bacteria employ NAD(P)-independent alcohol dehydrogenases (ADH) (Anthony, 1982). Instead of NAD(P), these enzymes use (PQQ), haem and/or F420 as cofactor. PQQ is typically a cofactor for the periplasmic methanol dehydrogenase (MDH) enzymes in methylotrophic Gram- negative bacteria (Duine and Frank, 1980). This is discussed in more detail in section 3.1.

2.3. NAD(P)-dependent alcohol dehydrogenases Three 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- or tetrameric quaternary structures and usually a subunit size of 43 kDa. Horse liver ADH is a Family I enzyme and has been studied in most detail. Family I ADHs show no, or relatively low, activities with methanol (Jörnvall et al., 1987; Reid and Fewson, 1994). Members of Family II are metal-independent, possess relatively short primary structures 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 a broad specificity and have diverse metabolic roles. There are no reports of their involvement in methanol oxidation in methylotrophic bacteria, however.

7 Chapter 1

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

Figure 2. Proposed quaternary structure of MNO of A. methanolica, based on electron microscopy and image processing analysis (Bystrykh et al., 1993a). A. top view, B. broad side view, C. narrow side view.

8 General introduction alcohol/acetylCoA dehydrogenase of Escherichia coli (Goodlove et al., 1989), and a gene of Entamoeba histolytica (Yang et al., 1994) of which only the C- terminal half of the deduced sequence shows similarity to ADH. No crystal structures of Family III ADHs thus far have been reported and only limited information is available about their secondary and tertiary structures. Electron microscopic studies and image analysis of five different members revealed decameric 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 Family III ADHs, since independent studies have demonstrated dimeric and tetrameric structures for two other members of Family III: ADH4 of Saccharomyces cerevisiae and ADHII of Zymomonas mobilis (Williamson and Paquin, 1987; Conway et al., 1987). Several Family III enzymes from aerobic methylotrophic Gram-positive bacteria are active with methanol (Table 1) and in vivo catalyze the oxidation of methanol to formaldehyde. These enzymes are reviewed in more detail in section 3.2 and are topic of further study in this thesis.

3. Methanol metabolism in bacteria Methylotrophic organisms use one-carbon compounds (e.g. methane, methanol, methylamine) as carbon source for growth. Several specific pathways for assimilation of C1 substrates have been elucidated in different aerobic methylotrophs (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 the ribulose biphosphate (RuBP)- or Calvin cycle. Yeasts follow the xylulose monophosphate (XuMP) or dihydroxyacetone cycle (not shown). The two other C1 assimilatory pathways identified in bacteria are the serine pathway (assimilating formaldehyde and CO2 ) and the ribulose monophosphate (RuMP) cycle (assimilating formaldehyde) (Figs. 1, 3). The RuMP cycle is energetically the most favourable of these pathways. The Gram-positive bacteria A. methanolica (Hazeu et 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 yields on methanol reported, stimulating considerable interest in possible biotechnological applications (production of single cell protein, enzymes, amino acids, vitamins) with these bacteria (Schendel et al., 1990; Dijkhuizen, 1993; Euverink, 1995).

9 Chapter 1

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

Enzyme Organism Family Gene Metals Subunits Reference

Ethanol:NDMA 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 by

dissimilating methanol to CO2 . Two different routes for methanol dissimilation are known, the linear pathway and the dissimilatory RuMP cycle (Dijkhuizen, 1993). The linear pathway involves the oxidation of methanol via formaldehyde and

formate to CO2 (Figs. 1, 3). Formaldehyde is a very toxic compound and its

10 General introduction

Figure 3. Dissimilatory and assimilatory pathways in C1 -utilizing bacteria (Levering, 1985). accumulation above 1 mM is lethal to the cell (Attwood and Quayle, 1984). The conversion of methanol to formaldehyde therefore requires a sensitive control and also accurate tuning with the next step, oxidation of formaldehyde to formate. In the dissimilatory RuMP cycle, ribulose 5-phosphate (RuMP) is coupled to formaldehyde, yielding hexulose-6-phosphate (H6P), and in a few steps converted to glucose 6-phosphate (G6P). The enzymes glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase subsequently produce NAD(P)H and carbon dioxide, finally also regenerating RuMP (Fig. 1).

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

11 Chapter 1

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

   MDH is an 22-tetrameric enzyme, with -subunits of 66 kDa and -subunits of 8.5 kDa in for instance Methylobacterium extorquens AM1 (Cox et al., 1992). The -subunit contains one molecule of the PQQ cofactor and one Ca2+ -ion in the . Crystal structures show eight radially arranged series of four -sheets in the main chain of the -subunit. This propeller like motif encloses PQQ in the active site 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 forming separate globular subunits.

Electrons derived from methanol oxidation are transferred to cytochrome cL (cytochrome c551i in Paracoccus denitrificans), which is different from other c-type cytochromes, since it is lacking the conserved features (Anthony, 1992b; Dales and

Anthony, 1995). Subsequently the electrons are transferred to cytochrome cH, cytochrome oxidase and finally to molecular oxygen (Fig. 4). This sequence of steps has not been confirmed in every organism studied thus far. Many redox mediators present in the periplasmic space are capable of reacting with each other. It is therefore not certain whether the electron transport chain involved is always linear, with more complex networks being possible as well. Mutants lacking specific cytochromes not always provide a clear phenotype, probably because some cytochromes can overtake each others function (Anthony, 1992b). The

12 General introduction concentration of the cytochromes in the periplasmic space is rather high, up to 0.5 mM (Anthony, 1986). The overall methanol oxidation system is very complex, requiring at least 30 different gene products. The biosynthesis of PQQ for instance already requires 8 gene products. The production of an active methanol dehydrogenase in the periplasm involves the following steps (Goodwin and Anthony, 1995): synthesis and export 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 of   two -units to form the 22-tetramer. Many of the genes involved have been characterized. Methanol oxidation in Gram-negative bacteria and the nomenclature of the genes involved have been reviewed by (Lidstrom et al., 1994). NAD(P)-independent ADHs, not active towards methanol, are somewhat more divergent. Some ADHs resemble MDH in solubility, subunit composition and having cytochrome c as primary electron acceptor (Anthony, 1992a; Schrover et al., 1993; Goodwin and Anthony, 1995). ADH of Comamonas testosteroni contains besides a PQQ molecule also a haem c molecule. It is therefore called a quinohaemoprotein (Groen et al., 1986; De Jong et al., 1995a, b). Similar soluble quinohaemoproteins have been identified in C. acidovorans, Pseudomonas putida and Rhodopseudomonas acidophila (Toyama, et al., 1995; Yasuda et al., 1996). Acetic acid bacteria (e.g. Acetobacter aceti, Gluconobacter suboxydans) contain a membrane-bound ADH, in quinohaemoprotein complexes, at the periplasmic side of the cytoplasmic membrane (Matsushita et al., 1992). This enzyme is important for the production of acetic acid from ethanol. It consists of three different components: the haem c and PQQ containing dehydrogenase, a trihaem cytochrome c containing subunit, and a third subunit with unidentified prosthetic groups and function (Matsushita et al., 1995). The third subunit is missing in the ADH isolated from A. polyoxogenes (Tayama et al., 1989). The various haem moieties are involved in the intermolecular electron transfer of ADH to ubiquinone, which is oxidized again by terminal ubiquinol oxidase, cytochrome o or a1 (Matsushita et al., 1992, 1996). Studies of ADH of A. aceti revealed that ADH contains a carboxyterminal extension with the haem c site. Similar to MDH, ADH also contains 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 have been 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.

13 Chapter 1

3.2. Enzymes involved in alcohol oxidation in Gram-positive bacteria In contrast to their Gram-negative counterparts, only a limited number of studies have been done on the biochemistry of primary alcohol oxidation in Gram-positive bacteria. A literature search for general ADHs characterized from Gram-positive bacteria resulted in a list of 30 enzymes (Table 1). Interestingly, all ADHs characterized in Gram-positive bacteria are NAD(P)-dependent and where studied, soluble, cytoplasmic proteins. The presence of ADHs active with methanol has been reported 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; Nešvera et 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 in sections 3.2.1 and 3.2.2, respectively. The limited knowledge available about ADHs in other Gram-positive bacteria is briefly summarized in section 3.2.3.

3.2.1. Amycolatopsis methanolica Amycolatopsis methanolica is a nocardioform actinomycete, showing the characteristic formation of a pseudomycelium on solid media, presence of spores and 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 actinomycete characterized, 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 be rather difficult to identify the enzymes involved in methanol oxidation in A. methanolica. Following its isolation from soil of New Guinea, Kato et al. (1975)

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

14 General introduction 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 with DCPIP and not with NAD. This activity was detected in methanol-growing cells, producing relatively large amounts of PQQ. It was suggested that n-MDH constituted a complex of methanol dehydrogenase, NAD-dependent formaldehyde dehydrogenase and NADH dehydrogenase activities (Fig. 5) (Duine et al., 1984a). Activities of n-MDH with DCPIP were stimulated by addition of NAD; the complex only showed activity with methanol, and not with ethanol. The n-MDH complex appeared rather instable, however, with no MDH activity remaining upon dissociation in its components. Other quinoproteins have been detected in A. methanolica but thus far no physiological functions could be assigned to any of them (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 MTT as an artificial electron acceptor. This enzyme system (MTT-ADH) showed activity with methanol and various other alcohols. MTT-ADH is considerably more stable than n-MDH, which allowed its more detailed characterization in subsequent experiments. Biochemical and mutant evidence showed that MTT-ADH also constitutes a protein complex of three different components, identified as methanol:NDMA oxidoreductase (MNO, see below) and so-called protein H (with MTT-dependent NADH dehydrogenase activity) and protein L (no separate activity identified) (Bystrykh et al., 1993b, 1997; Chapter 2-4). Following their purification an active MTT- ADH complex could be reconstituted by adding the various components together again (Bystrykh et al., 1997). MNO constitutes the first single protein with methanol dehydrogenase activity identified in A. methanolica (and in M. gastri) (Bystrykh et al., 1993a, b), using NDMA as artificial electron acceptor (Fig. 6). The molecular characterization Figure 6. Structure of and physiological role of this homo-decameric p-nitroso-N-N'-dimethylaniline. protein in A. methanolica is presented in Chapters 2-4 (Bystrykh et al., 1993a, b); the data show that the enzyme belongs to Family III of NAD(P)-dependent ADHs. It contains a tightly, but non-covalently bound NADP(H) which is redox-active and acts as cofactor. The associated proteins in the MTT-ADH complex most likely function in re-oxidation of the reduced NADPH

15 Chapter 1 cofactor. The in vivo electron acceptor for this system in A. methanolica remains to be identified. The n-MDH and MTT-ADH complexes may share one or more components, although n-MDH is highly specific for methanol and MTT-ADH shows a much broader substrate specificity (various primary and secondary alcohols). A separate ethanol:NDMA oxidoreductase (ENO) activity has been identified in A. methanolica (Van Ophem et al., 1993). This trimeric enzyme, with subunits of 39 kDa, contains a tightly bound NAD as cofactor and belongs to Family I. The enzyme is most active in methanol-grown cells, although it is not active with methanol. The in vivo role of ENO is unclear. Mutants lacking MNO, but still possessing 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 (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 + Wbox â ROCOOH + Wb 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 + DCPIPox â HCOOH + DCPIP red 5. Formaldehyde (MNO) (Bystrykh et al., 1993b; Chapters 2-4) â HCHO + HCHO CH3 OH + HCOOH

Each of these enzymes has been purified and characterized. Purified MNO shows high formaldehyde dismutase activities. The in vivo significance of this activity remains doubtful. Formaldehyde dismutase is known to increase the resistance of P. 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 sole carbon- and energy source (in formaldehyde-limited chemostat cultures) (Chapters

16 General introduction

3, 4). The strongest candidate for an in vivo role in formaldehyde degradation was FD-FAlDH. This enzyme is only found in methanol-grown cells and is specific for formaldehyde. The other formaldehyde-oxidizing enzymes also are active with higher aldehydes (Van Ophem et al., 1992b). However, a methanol-negative mutant of A. methanolica which lacked not only MNO but also FD-FAlDH and NA-AlDH could still grow on formaldehyde, displaying an increased level of DL-AlDH (Chapter 3). FD-FAlDH is a trimeric enzyme of 117 kDa, containing Zn2+ , and also 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 the NAD/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 factor remains unknown, but can be mimicked by high concentrations of methanol (Van Ophem et al., 1992b). This led to the hypothesis that the true substrate for FD-

FAlDH is a hemiacetal of methanol and formaldehyde (CH32 -O-CH OH) and that methylformate is the product, possibly an intermediate in an alternative formaldehyde oxidation pathway (Van Ophem et al., 1992a; Sakai et al., 1995). Methylformate or factor-formate would be the substrate for FEDH. Although methanol is not a substrate for FD-FAlDH, the enzyme is induced by growth on methanol. In contrast, ethanol did not induce FD-FAlDH, but NA-AlDH (Van Ophem and Duine, 1990a). This is a homotetramer of 200 kDa, with a broad substrate specificity, but not active with formaldehyde, although it is also induced during growth on methanol (Van Ophem and Duine, 1990a). FEDH and DL-AlDH are dye-linked enzymes; their in vitro activity is assayed with artificial electron acceptors since the biological acceptors are unknown. FEDH is a molybdoprotein, containing 1 Mo, 4 Fe, 3 or 4 S, and 1 FAD per enzyme molecule of 186 kDa (Van Ophem et al., 1992a; Kim et al., 1996). The enzyme is induced when growing on primary alcohols, with methanol and 1-hexanol as the best inducing substrates. The enzyme is active with aldehydes and formate esters (esters of formate and methanol or ethanol), but not with alcohols or formate separately. DL-AlDH is also a molybdoprotein, containing FAD, Fe and S. Just like FEDH and molybdoproteins in general, DL-AlDH consists of three different subunits, with a total mass of 160 kDa (87, 35 and 17 kDa) (Kim et al., 1996). In contrast to     FEDH, with subunit composition, DL-AlDH has an unique 2 structure. FEDH and DL-AlDH are two different enzymes, although their induction pattern and substrate specificity suggest a similar physiological role.

17 Chapter 1

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 methanolicus Bacillus methanolicus is a thermotolerant methylotrophic Gram-positive bacterium (Dijkhuizen et al., 1988; Arfman et al., 1992a).The organism was isolated for its capability to grow on methanol at elevated temperatures. B. methanolicus can grow on 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 processing revealed that B. methanolicus also possesses a decameric alcohol-oxidizing enzyme (Vonck et al., 1991). In contrast to MNO of A. methanolica, the methanol dehydrogenase of B. methanolicus (MDH) is active with NAD as coenzyme (Arfman et al., 1989). Cells grown under methanol-limiting conditions accumulate MDH up to 22 % of total soluble protein (Arfman et al., 1989), probably to overcome the poor affinity of MDH for methanol. MDH contains Mg2+ and Zn 2+ instead of iron (Vonck et al., 1991). MDH contains a tightly, but non-covalently bound NAD; further studies showed that this NAD(H) is redox-active and functions as a cofactor (Arfman et al., 1997; Chapter 5). The mdh gene has been characterized, resulting in classification of MDH as a Family III enzyme (De Vries et al., 1992). B. methanolicus possesses a special activator protein, consisting of two subunits of 27 kDa, which stimulates the relatively low activity of MDH towards methanol (Arfman et al., 1991). This stimulation is Mg2+ -dependent and results in a 40-fold increase in MDH turnover rate. In Chapters 5 and 6 we provide evidence that the activator 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 of the MutT family (Koonin, 1993). The E. coli MutT protein is a Mg2+ -dependent triphosphatase, hydrolyzing 8-oxo-dGTP as preferred substrate. The nucleotide 8- oxo-dGTP can pair with cytosine as well as adenine, causing errors in DNA replication. MutT degrades 8-oxo-dGTP into 8-oxo-dGMP and pyrophosphate, thereby preventing these mutations to occur (Bullions et al., 1994). The mechanism of the B. methanolicus activator protein remains to be studied in more detail. In vivo this protein may have an important physiological role, contributing to the control of MDH activity. MDH is synthesized in abundant amounts, making its control a delicate problem since accumulation of formaldehyde, the product of the MDH reaction, is lethal to the cell (Arfman et al., 1992b).

18 General introduction

3.2.3. Other Gram-positive bacteria ADHs have been identified and characterized in a few other Gram-positive bacteria. Three different ADHs have been isolated from three different Bacillus stearothermophilus strains. ADH2334 of strain DSM 2334, an obligately aerobic organism, possesses activity with methanol (Km 20 mM). The enzyme is substrate 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 a thermostable enzyme, inactive with methanol, insensitive to substrate inhibition, and involved in ethanol production. The even more heat stable ADH-hT, isolated of strain NCIMB 12403, is also inactive with methanol and involved in ethanol production (Guagliardi et al., 1996). Sequence data show an high degree of identity, but biochemical and immunological data clearly suggest that these three enzymes are 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 Family I and have subunit sizes of approximately 36 kDa. A single member of Family III ADHs 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 assimilates methanol carbon via the serine pathway (Bastide et al., 1989). Methanol dehydrogenase activity could be detected with phenazine methosulphate as artificial electron acceptor. Brevibacterium methylicum assimilates methanol carbon via the RuMP cycle. This organism possesses NAD-dependent MDH activity (Nešvera et al., 1991). The methylotrophic actinomycete strain 381 was reported to possess an n-MDH activity (Eshraghi et al., 1990), comparable to A. methanolica (Duine et al., 1984a). The further characterization of these methanol oxidizing enzymes has not been reported. Most ADHs have been characterized from various Clostridium species. Much effort has been devoted to the characterization of these enzymes, mainly because of their involvement in production of the commercially interesting butanol and 2- propanol. C. acetobutylicum contains several different ADHs. First of all there are two butanol dehydrogenases (BDHI and BDHII). These isoenzymes prefer NADH above NADPH as coenzyme (Walter et al., 1992; Petersen et al., 1994), although Chen (1995) reported that this preference is pH-dependent. The gene adh1 codes for a different NADPH-dependent ADH involved in the production of butanol and ethanol (Youngleson et al., 1989), while aad codes for a bifunctional enzyme (Nair et al., 1994). The N-terminal part of the protein shows similarity with aldehyde

19 Chapter 1 dehydrogenases, while the C-terminal half of the protein encodes an alcohol dehydrogenase (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 for protein characterization make it difficult to establish clear resemblances and differences between the ADHs of Clostridia. Data presented thus far indicate that all primary ADHs of Clostridium species belong to Family III (Table 1). This also includes the butyraldehyde dehydrogenase involved in succinate degradation by C. kluyveri, which is part of a bifunctional protein as also reported for the alcohol/aldehyde dehydrogenase of C. acetobutylicum(Söhling and Gottschalk, 1996; Nair et al., 1994). Thermoanaerobacter ethanolicus contains a primary ADH with a temperature optimum of 85°C (Bryant et al., 1988; Burdette and Zeikus, 1994). The enzyme is a homotetramer of 41.5 kDa subunits and contains zinc. It has been hypothesized that a secondary ADH, also active with ethanol and propanol, is responsible for most of the ethanol production, while the primary ADH is involved in the ethanol consumption (Bryant et al., 1988). The catalytic efficiency of the primary ADH for NADH oxidation is eight times higher than for NADPH oxidation and the reduction of NADP is 30 times more efficient than NAD reduction. Corresponding efficiencies for acetaldehyde reduction and ethanol oxidation suggest that the primary ADH can control the NADH and NADPH pools by cycling between acetaldehyde and ethanol (Lovitt et al., 1988; Burdette and Zeikus, 1994). A comparable situation has been found in Thermoanaerobacter brockii (Lamed and Zeikus, 1980, 1981). No sequence information is available for these enzymes; in view of their molecular weights and presence of zinc they are either Family I or Family III ADHs. Several Family II ADHs have been identified in the genus Streptomyces. One of these, 20-hydroxysteroid dehydrogenase, has been characterized from S. hydrogenans (Marekov et al., 1990). A second one, the actIII gene product, has been found in S. coelicolor A3(2) and is involved in biosynthesis of the polyketide antibiotic actinorhodin (Hallam et al., 1988; Baker, 1990). Family II ADHs may be more widespread in Streptomyces. Antibodies raised against Drosophila ADH (Family II) showed no cross-reactivity with the 20-hydroxysteroid dehydrogenase but 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 later growth phase, indicating its possible involvement in the production of secondary

20 General introduction metabolites or in morphological differentiation. Since the actIII-product was missing in S. lividans its 28-30 kDa protein should be another Family II ADH. ADH of Mycobacterium tuberculosis var. bovis (BCG) has been partially purified and characterized as a dimer with subunits of 37.5 kDa, containing zinc (De Bruyn et al., 1981). Highest affinity was observed with butyraldehyde (125 µM); the affinity for butanol was 220 mM. Based on the deduced amino acid sequence of adh the 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 primary alcohols, but not with methanol (Ashref and Murrell, 1990). This NAD-dependent enzyme is a dimer with subunits of 42 kDa. R. erythropolis and A. methanolica contain 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; Chapters 2-4). MNO has a key function in methanol oxidation in the latter two methylotrophic bacteria, but Rhodococcus sp. NI86/21 is unable to grow on methanol. The ThcE protein is induced during growth with thiocarbamate and is thought to be involved in the biodegradation of this and other herbicides (Nagy et al., 1995). MTT-dependent alcohol dehydrogenase (MTT-ADH) activities have been reported 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 of a complex of at least 3 proteins including MNO (see above). MNO has also been identified 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-binding Alcohol dehydrogenase enzymes oxidize an alcohol group of their substrates by transfer of a hydride-ion from the carbon atom that binds the hydroxyl group to the oxidized form of their coenzymes or cofactors. In addition, a proton is removed from the alcohol hydroxyl group (Fig. 7). During dehydrogenation this hydride-ion

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

21 Chapter 1 and proton are directly transferred from substrate to coenzyme/cofactor. These enzymes, therefore, not only must recognize and bind substrate and for instance NAD(P), but also must position them in the active site, sufficiently close and in the correct orientation to allow direct hydrogen transfer to take place. The dinucleotide- binding domains of various dehydrogenases have very similar three-dimensional structures, 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 30-35 residues 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

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

22 General introduction

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 and Tooze, 1991). Nevertheless, there are strong stereochemical constraints at specific positions in the polypeptide chain of the Rossmann-fold that must be respected to preserve its structure and function. These invariant key residues provide a fingerprint to predict dinucleotide-binding regions in proteins of known amino acid sequence but with unknown three-dimensional structure (Wierenga et al., 1986). Most recognizable is the Gly rich GXGXXG motif (X, variable amino acid). An analysis of 133 sequences of FAD- or NAD-dependent proteins (Nishiya and Imanaka, 1996) confirmed the presence of the conserved GXGXXG motif. In addition, in 56 sequences (42 %) the second position was occupied by a Gly or Ala residue, resulting in G(GA)GXXG. Large, acidic, basic or aromatic amino acids did not appear so often at this second position. The GXGXXG motif, together with six conserved hydrophobic amino acids, enables formation of a  fold, also positioning an Asp or Glu residue that forms a hydrogen bond with the 2'-OH of the ADP-moiety of NAD and FAD (Figs. 8, 9). The 2'-OH position in NADP(H) is occupied by a phosphate group; in an NADP-binding domain the Asp or Glu residue has 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

23 Chapter 1 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 coenzyme specificity of NADP-dependent glutathione reductase of E. coli towards NAD (Scrutton et al., 1990; Mittl et al., 1993, 1994). Single mutations did cause a shift in coenzyme specificity of this protein but these mutants were catalytically less efficient than the wild type enzyme. Not only amino acid side-chains but also main chain NH-groups may form hydrogen-bonds with the coenzyme, which diminishes the effects of point mutations (Baker et al., 1992). Although the GXGXXG motif is well conserved, some variations do occur (Lesk, 1995). In 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 and the -helix. Also 20-hydroxysteroid dehydrogenase of S. hydrogenans contains an insertion in the loop, yielding the sequence pattern which is conserved in ADHs of Family II: GXXXG(A)XG (Ghosh et al., 1991; Lesk, 1995). In NADP-dependent 6-phophogluconate dehydrogenase of sheep and dihydropteridine reductase of rat the position of the second Gly is occupied by an Ala residue, which causes a different interaction between enzyme and coenzyme (Shahbaz et al., 1987; Lesk, 1995). Further variations in the Gly rich motif for NADP-binding (GXGXGXXPF) and for NAD-binding (GGXGXFP), still allowing formation of -folds, were found in 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 proteins did show some sequence similarity, only a Ser residue was fully conserved. This group includes the human proteins NADPH-cytochrome b245 and NADH- cytochrome b5 reductase, the rat proteins NADPH-cytochrome P-450 reductase and NADPH-nitric oxide synthase (Bredt et al., 1991), NADH-nitrate reductase of tomato, ferredoxin-NADP reductase (FNR) of spinach, NADPH-sulphite reductase of Salmonella typhimurium, and NADPH-cytochrome P-450 reductase from Bacillus megaterium (Segal et al., 1992). The atomic structure of FNR demonstrated an antiparallel ß-barrel core and a single -helix as unusual binding domain for the pyrophosphate of FAD (Karplus et al., 1991). The actual interactions between FAD and the protein are with Arg, Ser, Tyr residues, and the peptide amides at the aminoterminal end of the -helix. The presence of a Gly residue again allows a close approach of the helix to the pyrophosphate group. A total of 6 peptide segments involved in FAD and NADP-binding could be identified; these are conserved in enzymes of the FNR-family. Also the spacing between these segments

24 General introduction is the same, this conservation is significant, although the separate segments are too short to indicate individual similarities (Karplus et al., 1991). The NAD(P)-binding domains in ADHs of Families I and II generally can be identified straightforwardly, when searching for the GXGXXG fingerprint. Only a few members of Family III ADHs possess this classical dinucleotide-binding domain (De Vries et al., 1992; Chapter 4). Chapter 6 describes the characteristics of site-directed mutants of MDH of B. methanolicus, allowing identification of a new NAD(P)(H)-binding domain in members of Family III ADHs.

5. Nicotinoproteins NAD(P) functions as a coenzyme for a large variety of dehydrogenase enzymes, receiving or donating electrons depending on the specific reaction catalyzed and the reaction conditions. The cytosolic NAD(P)(H) can be oxidized or reduced elsewhere in the cell, e.g. by NAD(P)H dehydrogenase in the cytoplasmic membrane. In recent years a limited number of so-called nicotinoproteins, containing tightly but noncovalently 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 bound to the enzyme during . An external electron donor or acceptor subsequently may reduce or oxidize the cofactor in situ. Zymomonas mobilis for instance couples the 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:oxaloacetate oxidoreductase of Veillonella alcalescens (Allen, 1966, 1982), UDP-galactose 4- epimerase from E. coli (Bauer et al., 1992) and formaldehyde dismutase of P. putida F61 (Kato et al., 1986). The latter enzyme serves to strongly increase formaldehyde resistancy of the cells, coupling oxidation of one formaldehyde molecule to formate with reduction of a second formaldehyde molecule to methanol (Kato et al., 1986). Studies of methanol-utilizing Gram-positive bacteria have resulted in identification 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 physiological role appears to be in oxidation of primary alcohols (Chapters 3, 4). The in vivo electron acceptor for MNO remains to be identified. Also MDH of B. methanolicus

25 Chapter 1 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 MDH and 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 thesis Aim of this thesis was to investigate the physiology and biochemistry of oxidation of methanol (and other primary alcohols) in Gram-positive bacteria. Most of the current knowledge of methanol oxidation is based on studies with Gram-negative methylotrophs and the number of detailed studies of their Gram-positive counterparts is limited. The methanol-oxidizing systems in Gram-negative bacteria are located in the periplasm and use PQQ as cofactor. This prompted questions about the nature and location of these systems in Gram-positive organisms which lack a clear periplasmic space and generally do not possess PQQ. The studies described in this thesis focussed on two methanol-utilizing bacteria, the actinomycete A. methanolica and the thermotolerant bacterium B. methanolicus. Previous studies suggested that these bacteria employ novel ADH protein complexes in the metabolism of lower primary aliphatic alcohols (Duine et al., 1984a, b; Van Ophem et al., 1991; Arfman et al., 1991). Interestingly, several reports also provide evidence for the presence of PQQ in A. methanolica; its physiological role has remained unclear, however (Hazeu et al., 1983; Duine et al., 1984a; Van Ophem and Duine, 1990b). Preliminary evidence also indicated that these novel enzymes occur more widespread in actinomycetes. Various approaches were followed, involving application of (at random and site-directed) mutagenesis, biochemical techniques (protein purification and characterization) and molecular techniques (gene cloning, site-directed mutagenesis), to characterize these enzyme systems in more detail. Chapter 1 reviews current knowledge of microbial primary alcohol oxidation, with emphasis on Gram-positive bacteria. The purification and characterization of an A. methanolica enzyme with high formaldehyde dismutase activity, but also oxidizing methanol and other primary alcohols, is described in Chapter 2 (Bystrykh et al., 1993a, b). This methanol:NDMA oxidoreductase (MNO) shows no activity with coenzyme NAD(P) but otherwise shares similarities with the previously described NAD-dependent methanol dehydrogenase (MDH) of B. methanolicus (Arfman et al., 1989; Vonck et al., 1991). Both enzymes are for instance decameric nicotinoproteins containing NAD(P)(H) cofactors (Bystrykh et al., 1993a). MNO appears to be part of a protein complex with proteins H and L, showing MTT-

26 General introduction dependent ADH activity (Bystrykh et al., 1997). The characterization of A. methanolica mutants unable to grow on methanol provided clear evidence that MNO and protein H are essential for the metabolism of primary alcohols in general (Chapter 3). Cloning and characterization of the MNO-encoding gene allowed construction of the A. methanolica derivative strain MDM2 with a disrupted mno gene (single cross-over). Characterization of this mutant strain confirmed that loss of MNO resulted in complete failure to grow on primary alcohols; growth on formaldehyde remained possible, however. Sequence alignments showed that MNO (and MDH) are members of the steadily growing Family III of NAD(P)-dependent ADHs (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). Biochemical studies of the reaction cycle of the B. methanolicus MDH demonstrated that its tightly, but non-covalently, bound NAD is redox-active and remains bound during catalysis. An exogenous, coenzyme NAD molecule, is able to re-oxidize the reduced NADH cofactor, resulting in relatively low NAD-MDH activities. This step is facilitated by a B. methanolicus activator protein of 50 kDa (Arfman et al., 1991) which strongly stimulates MDH turnover in the presence of NAD and Mg2+ -ions. Alignments of the full length sequences of the 24 Family III ADHs currently known initially failed to identify the classical dinucleotide-binding fold for NAD(P)(H). Three unique, conserved sequence motifs, were detected in these proteins, however. Following site-directed mutagenesis of MDH (Chapter 6), evidence was obtained for the involvement of several amino acids in one of these motifs in NAD cofactor and coenzyme-binding. This conserved motif thus may be part of a new NAD(P)-binding fold in Family III ADHs.

References References are listed on pages 127 - 136.

27