224
S-6-5
Biochemistry Quinone
and Physiology of Pyrroloquinoline and Quinoprotein
O .ADACHI,1
1 Department Yamaguchi Department University, I.
of
K.MATSUSHITA,1
Biological
University, 1677-1, of Biotechnology, Suita 564 (Japan)
Chemistry Yoshida, Faculty
Dehydrogenases and
,
M.AMEYAMA2
Faculty of Agriculture, Yamaguchi 753 (Japan)2 of Engineering, Kansai
INTRODUCTION
Metabolic significance of pyrroloquinoline quinone (PQQ) has been made clear in these several years and more than 10 species of dehydrogenases carrying PQQ (quinoprotein) as the prosthetic group have been identified in Gram-negative bacteria. Occurrence of many species of quinoprotein dehydrogenase has been indicated in acetic acid bacteria and their biological functions in relation to bioenergetics and oxidative fermentation have been elucidated (1). Thus, PQQ has been evaluated as the third coenzyme following pyridine nucleotide and flavin in biological oxidoreduction. Furthermore, PQQ nas been detected in many biological sources from procaryotes to eucaryotes and some interesting physiological functions of PQQ, a potent growth stimulant for cell growth, have also been indicated (2-6). In the meantime, PQQ has become commercially available (5,7). In this paper, biochemical and physiological functions of PQQ in acetic acid bacteria and others are summarized. II.
QUINOROTELNS
ACID
BACTERIA
INVOLVED
IN OXIDATIVE,
FERMENTATION
OF
ACETIC
Acetic acid bacteria are well known as a vinegar producer, and divided into two genera Acetobacter and Gluconobacter. Acetic acid bacteria are able not only to oxidize etnanol to acetic acid , but also able to oxidize glucose, 9luconic acid, glycerol, sorbitol and so on. These sugar oxidation reaction Nave been called as "oxidative fermentation", the study of which has a long histroy since Louis Pasteur but the mechanism of which has been elucidated only recently. Tne enzyme involved in "oxidative fermentation" are all tightly bound to the outer surface of the cytoplasmic membrane of the organism, and can be divided into two groups, quinoproteins and flavoproteins. The quinoproteins include glucose (GDH), alcohol (ADH), aldehyde, fructose, and glycerol dehydrogenases, while the others containing covalently bound fiavin are gluconate, 2-ketogluconate and sorbitul dehydrogenases. These sugar-oxidizing dehydrogenases work coupling with the respiratory chain located in tne cytoplasmic membrane of acetic acid bacteria. Tue respiratory chain of acetic acid bacteria is somewhat different between Gluconobacter and Acetobacter. A. aceti has a simple respiratory chain brancning at the terminal end with cytochrome o and cytochrome al, which are mutually changed by the growth conditions, respectively, static and shaking cultures (8). Since the both terminal oxidases are functioning as ubi(duinol oxidase, the respiratory chain would be expected as shown in Fig.1.
O .ADACHI
Acetobacter
Fig.1.
Respiratory
et
al.
225
acet
Chains
Gluconobacter
of
A.
aceti
and
G.
suboxydans
suboxydans.
Whereas, G. suboxydans respiratory chain has a characteristically high concentrations of cytochrome c and cytochrome o as the terminal ubiquinol oxidase. The cytochrome o content is not changed. The increase of cytochrome c in the respiratory cahin is accompanied by the decrease of cyanidesensitivity; the cells grown at lower extracellular pH exhibits cyanide-resistant bypass compared with the cells grown at high pH (9). Furthermore, the respiratory chain of G. suboxydans var a, ADH-deficient strain, naturally occurred, lacks both characters, cyanide-resistant respiration and high cytochrome c content, which could be restored by reconstituting purified ADH into the membranes. Thus, the respiratoy chain of G. suboxydans can be depicted as shown in Fig. 1. III,
PUQ, A GROWTH STIMULATING SUBSTANCE FOR MICROORGANISMS It is well-known phenomenon that the growth of microorganisms is stimulated markedly by the addition of various kinds of naturally occurring substances to the culture medium. In the course of investigations on the growth of acetic acid bacteria, it was observed that the growth of Acetobacter species, which show no appreciable nutritional requirements, was stimulated markedly by the addition of yeast extract to a synthetic medium in which generally known growth factors of vitamins, amino acids or nucleic acids were fully supplied (10). As to strains of the genus Gluconobacter, on the other hand, some species have been shown to require vitamins or others. Addition of yeast extract to a complete synthetic medium for such Gluconobacter strains further stimulated the growth of the organisms. A similar growth stimulating effect to that observed with yeast extract was also found with other naturally occurring substances used for culture ingredients for microorganisms (4). It is also generally accepted that the growth simulation observed with such naturally occurring substances is not restricted to only acetic acid bacteria but also in a variety of microbial genera. The growth stimulating substance in these materials and culture broths of Escherichia coli, methylotrophs or pseudomonads has been isolated and identified to be P or more probably to be PQQ-adducts (3,4,11). Our earlier observations that E. call produce PQQ more or less in the culture medium (3) has been supported by current reports that E. coli produces holo-upH (12,13). Two types of growth stimulating effect of PQQ for microorganisms has been proved (Fig.2). The type I PQQ effect
Symposium (6)
226
Vitamin
B6 and PQQ
was observed in a symbiotic polyvinyl alcohol (PVA) degradation in which one species of Pseudomonas excretes PQQ which in turn enables the other species to grow on PVA (11). The latter can produce a carbon source for the former only when PQQ is supplied. Thus,PQQ is regarded as the essential growth factor of the PVAdegrading bacteria when grown on PVA but not on other common carbon sources. The type II growth stimulation by PQQ is characterized by a marked reduction of the lag phase in the presence of PQQ or PQQadducts (2,5,6). In this case, the subsequent growth rate at the exponential phase and the total cell yield at the stationary phase are not affected at all. Unlike the former type, PQQ is not always an essential growth factor and normal cell growth can be seen even in the absence of exogenous PQQ alter a relatively prolonged lag time. This type of PQQ effect was discovered in the course of searching for a growth stimulating substance in yeast extract for acetic acid bacteria. The same growth stimulating effect was observed with E. coli, pseudomonads and others, when a trace amount of PQQ or PQQ-adduct was given to their synthetic medium. Thus, PQQ effect has been proved to be a universal phenomenon in the initial stage of cell proliferation, sounding that PQQ has a fundamental importance in basic cell pnysiology. In future study, it has to be focused to see wnich part of biological steps is stimulated by PQQ.
Culture
Fig.2.
Growth
Stimulating Effects
period
(hr)
of PQQ
for MIcroorganisms.
PQQ-adduct formation reauily occurs during extraction and isolation procedure for PQQ from the naturally occurring materials or PQQ-cnromopnore from quinoproteins. Such adduct must give a low PQQ estimation than Lheoretically expected. PQQ may exist in vivo as free form or associated to a guinoprotein or a r carrier procein but it seems ai f cut to isolate PQQ as free form, except for PQQ production by methylotrophs (7,5). In other words, a small amount of in vivo tends to react with amino compounds to form i,,e-adducts auring hanalings ano b.comes less active as tne prosthetic group. With such PQQ-adducts, content cannot be estimaten exactly witn a guinoprotein glucose de hydrogenase whereas growth stimulating activity can be estimated as much the some as free PQQ. From these observations mentioned above, PQQ activity snould be followed by both criteria, coenzyme activity for quinoproteins and growth stimulating activity for microorganisms.
O .ADACHI
et
al.
227
REFERENCES
(1)
Ameyama, Bacteria.
(2)
Ameyarna, M., Shinagawa, E., Matsushita, K., and Adachi, O. (1984): Growth Stimulation of Microorganisms by Pyrroloquinoline Quinone. Agric. Biol. Chem., 48, 29092911. Ameyama, M., Shinagawa, E., Matsushita, K. and Adachi, O. (1984): Growth Stimulating Substance for Microorganisms Produced by Escherichia coli Causing the Reduction of the Lag Phase in Microbial Growth and Identity of the Substance with Pyrroloquinoline Quinone. Agric. Biol. Chem., 48, 3099-3107. Ameyama, M., Shinagawa, E., Matsushita, K. and Adachi, O. (1985): Growth Stimulating Activity for Microorganisms in Naturally Occurring Substance and Partial Characterization of the Substance for the Activity as Pyrroloquinoline Quinone. Agric. Biol. Chem., 49, 699-709. Ameyama, M., Matsushita, K., Shinagawa, E. and Adachi, O. (1988): Pyrroloquinoline Quinone; Excretion by Methylotrophs and Growth Stimulation for Microorganisms. BioFactors, 1, 51-53.
(3)
(4)
(5)
(6)
(7)
M. (1988): Biochemical Nippon Nogeikagaku Kaishi,
Studies 62,
on Acetic 1185-1193.
Acid
Adachi, O., Okamoto, K., Shinagawa, E., Matsushita, K. and Ameyarna, M. (1988): Growth Stimulating Activity for Microorganisms of an Adduct formed with Pyrroloquinoline Quinone and Amino Acid. BioFactors, 1, 251-254.
Ameyama, M., Hayashi, M., Matsushita, K., Shinagawa, E. and Adachi, O. (1984): Microbial Production of Pyrroloquinoline Quinone. Agric. Biol. Chem., 46, 561-565. (8) Matsushita, K., Shinagawa, E., Adachi, O. and Ameyama, M. (1990): Cytochrome al of Acetobacter aceti is a Cytochrome ba Functioning as Ubiquinol Oxidase. Proc. Natl. Acad. Sci. USA., 87, 9863-9867. (9) Matsushita, K., Nagatani, Y., Shinagawa, E., Adachi, O. and Ameyama, M. (1989): Effect of Extracellular pH on the Respiratory Chain and Energetics of Gluconobacter suboxydans. Agric. Biol. Chem., 53, 2895-2902. (10) Ameyama, M. and Kondo, K. (1966): Carbohydrate Metabolism by the Acetic Acid Bacteria. Part V. On the Vitamin Requirements for the Growth. Agric. Biol. Cnem., 30, 203211. (11) Snimao, M., Yamamoto, H., Ninomiya, K., Kato, N., Adachi, O. Ameyama, M. and Sakazawa, C. (1934): Pyrroloquinoline tlinone as an Essential Growtii Factor forQ a Poly(vinylalcohol)-degrading Symbiont, Pseudomonas sp. VM15C. Agric. Biol. Chem., 48, 2873-2876. (12) Bouvet, O. M. M., Pascal, L. and Grimont, P. A. D. (1939): Taxonomic Diversity of the D-Glucose Oxidation Pathway in the Enterobacteriaceae. Intl. J. Syst.Bacteriol., 39, 6167. (13) Biville, F., Turline, E. and Gasser,. (1991): Mutants of Escherichia coli Producing Pyrroloquinoline Quinone. J. Gen. Microbial., in press.
S-6-5
Biochemistry Quinone
and Physiology of Pyrroloquinoline and Quinoprotein
O .ADACHI,1
1 Department Yamaguchi Department University, I.
of
K.MATSUSHITA,1
Biological
University, 1677-1, of Biotechnology, Suita 564 (Japan)
Chemistry Yoshida, Faculty
Dehydrogenases and
,
M.AMEYAMA2
Faculty of Agriculture, Yamaguchi 753 (Japan)2 of Engineering, Kansai
INTRODUCTION
Metabolic significance of pyrroloquinoline quinone (PQQ) has been made clear in these several years and more than 10 species of dehydrogenases carrying PQQ (quinoprotein) as the prosthetic group have been identified in Gram-negative bacteria. Occurrence of many species of quinoprotein dehydrogenase has been indicated in acetic acid bacteria and their biological functions in relation to bioenergetics and oxidative fermentation have been elucidated (1). Thus, PQQ has been evaluated as the third coenzyme following pyridine nucleotide and flavin in biological oxidoreduction. Furthermore, PQQ nas been detected in many biological sources from procaryotes to eucaryotes and some interesting physiological functions of PQQ, a potent growth stimulant for cell growth, have also been indicated (2-6). In the meantime, PQQ has become commercially available (5,7). In this paper, biochemical and physiological functions of PQQ in acetic acid bacteria and others are summarized. II.
QUINOROTELNS
ACID
BACTERIA
INVOLVED
IN OXIDATIVE,
FERMENTATION
OF
ACETIC
Acetic acid bacteria are well known as a vinegar producer, and divided into two genera Acetobacter and Gluconobacter. Acetic acid bacteria are able not only to oxidize etnanol to acetic acid , but also able to oxidize glucose, 9luconic acid, glycerol, sorbitol and so on. These sugar oxidation reaction Nave been called as "oxidative fermentation", the study of which has a long histroy since Louis Pasteur but the mechanism of which has been elucidated only recently. Tne enzyme involved in "oxidative fermentation" are all tightly bound to the outer surface of the cytoplasmic membrane of the organism, and can be divided into two groups, quinoproteins and flavoproteins. The quinoproteins include glucose (GDH), alcohol (ADH), aldehyde, fructose, and glycerol dehydrogenases, while the others containing covalently bound fiavin are gluconate, 2-ketogluconate and sorbitul dehydrogenases. These sugar-oxidizing dehydrogenases work coupling with the respiratory chain located in tne cytoplasmic membrane of acetic acid bacteria. Tue respiratory chain of acetic acid bacteria is somewhat different between Gluconobacter and Acetobacter. A. aceti has a simple respiratory chain brancning at the terminal end with cytochrome o and cytochrome al, which are mutually changed by the growth conditions, respectively, static and shaking cultures (8). Since the both terminal oxidases are functioning as ubi(duinol oxidase, the respiratory chain would be expected as shown in Fig.1.
O .ADACHI
Acetobacter
Fig.1.
Respiratory
et
al.
225
acet
Chains
Gluconobacter
of
A.
aceti
and
G.
suboxydans
suboxydans.
Whereas, G. suboxydans respiratory chain has a characteristically high concentrations of cytochrome c and cytochrome o as the terminal ubiquinol oxidase. The cytochrome o content is not changed. The increase of cytochrome c in the respiratory cahin is accompanied by the decrease of cyanidesensitivity; the cells grown at lower extracellular pH exhibits cyanide-resistant bypass compared with the cells grown at high pH (9). Furthermore, the respiratory chain of G. suboxydans var a, ADH-deficient strain, naturally occurred, lacks both characters, cyanide-resistant respiration and high cytochrome c content, which could be restored by reconstituting purified ADH into the membranes. Thus, the respiratoy chain of G. suboxydans can be depicted as shown in Fig. 1. III,
PUQ, A GROWTH STIMULATING SUBSTANCE FOR MICROORGANISMS It is well-known phenomenon that the growth of microorganisms is stimulated markedly by the addition of various kinds of naturally occurring substances to the culture medium. In the course of investigations on the growth of acetic acid bacteria, it was observed that the growth of Acetobacter species, which show no appreciable nutritional requirements, was stimulated markedly by the addition of yeast extract to a synthetic medium in which generally known growth factors of vitamins, amino acids or nucleic acids were fully supplied (10). As to strains of the genus Gluconobacter, on the other hand, some species have been shown to require vitamins or others. Addition of yeast extract to a complete synthetic medium for such Gluconobacter strains further stimulated the growth of the organisms. A similar growth stimulating effect to that observed with yeast extract was also found with other naturally occurring substances used for culture ingredients for microorganisms (4). It is also generally accepted that the growth simulation observed with such naturally occurring substances is not restricted to only acetic acid bacteria but also in a variety of microbial genera. The growth stimulating substance in these materials and culture broths of Escherichia coli, methylotrophs or pseudomonads has been isolated and identified to be P or more probably to be PQQ-adducts (3,4,11). Our earlier observations that E. call produce PQQ more or less in the culture medium (3) has been supported by current reports that E. coli produces holo-upH (12,13). Two types of growth stimulating effect of PQQ for microorganisms has been proved (Fig.2). The type I PQQ effect
Symposium (6)
226
Vitamin
B6 and PQQ
was observed in a symbiotic polyvinyl alcohol (PVA) degradation in which one species of Pseudomonas excretes PQQ which in turn enables the other species to grow on PVA (11). The latter can produce a carbon source for the former only when PQQ is supplied. Thus,PQQ is regarded as the essential growth factor of the PVAdegrading bacteria when grown on PVA but not on other common carbon sources. The type II growth stimulation by PQQ is characterized by a marked reduction of the lag phase in the presence of PQQ or PQQadducts (2,5,6). In this case, the subsequent growth rate at the exponential phase and the total cell yield at the stationary phase are not affected at all. Unlike the former type, PQQ is not always an essential growth factor and normal cell growth can be seen even in the absence of exogenous PQQ alter a relatively prolonged lag time. This type of PQQ effect was discovered in the course of searching for a growth stimulating substance in yeast extract for acetic acid bacteria. The same growth stimulating effect was observed with E. coli, pseudomonads and others, when a trace amount of PQQ or PQQ-adduct was given to their synthetic medium. Thus, PQQ effect has been proved to be a universal phenomenon in the initial stage of cell proliferation, sounding that PQQ has a fundamental importance in basic cell pnysiology. In future study, it has to be focused to see wnich part of biological steps is stimulated by PQQ.
Culture
Fig.2.
Growth
Stimulating Effects
period
(hr)
of PQQ
for MIcroorganisms.
PQQ-adduct formation reauily occurs during extraction and isolation procedure for PQQ from the naturally occurring materials or PQQ-cnromopnore from quinoproteins. Such adduct must give a low PQQ estimation than Lheoretically expected. PQQ may exist in vivo as free form or associated to a guinoprotein or a r carrier procein but it seems ai f cut to isolate PQQ as free form, except for PQQ production by methylotrophs (7,5). In other words, a small amount of in vivo tends to react with amino compounds to form i,,e-adducts auring hanalings ano b.comes less active as tne prosthetic group. With such PQQ-adducts, content cannot be estimaten exactly witn a guinoprotein glucose de hydrogenase whereas growth stimulating activity can be estimated as much the some as free PQQ. From these observations mentioned above, PQQ activity snould be followed by both criteria, coenzyme activity for quinoproteins and growth stimulating activity for microorganisms.
O .ADACHI
et
al.
227
REFERENCES
(1)
Ameyama, Bacteria.
(2)
Ameyarna, M., Shinagawa, E., Matsushita, K., and Adachi, O. (1984): Growth Stimulation of Microorganisms by Pyrroloquinoline Quinone. Agric. Biol. Chem., 48, 29092911. Ameyama, M., Shinagawa, E., Matsushita, K. and Adachi, O. (1984): Growth Stimulating Substance for Microorganisms Produced by Escherichia coli Causing the Reduction of the Lag Phase in Microbial Growth and Identity of the Substance with Pyrroloquinoline Quinone. Agric. Biol. Chem., 48, 3099-3107. Ameyama, M., Shinagawa, E., Matsushita, K. and Adachi, O. (1985): Growth Stimulating Activity for Microorganisms in Naturally Occurring Substance and Partial Characterization of the Substance for the Activity as Pyrroloquinoline Quinone. Agric. Biol. Chem., 49, 699-709. Ameyama, M., Matsushita, K., Shinagawa, E. and Adachi, O. (1988): Pyrroloquinoline Quinone; Excretion by Methylotrophs and Growth Stimulation for Microorganisms. BioFactors, 1, 51-53.
(3)
(4)
(5)
(6)
(7)
M. (1988): Biochemical Nippon Nogeikagaku Kaishi,
Studies 62,
on Acetic 1185-1193.
Acid
Adachi, O., Okamoto, K., Shinagawa, E., Matsushita, K. and Ameyarna, M. (1988): Growth Stimulating Activity for Microorganisms of an Adduct formed with Pyrroloquinoline Quinone and Amino Acid. BioFactors, 1, 251-254.
Ameyama, M., Hayashi, M., Matsushita, K., Shinagawa, E. and Adachi, O. (1984): Microbial Production of Pyrroloquinoline Quinone. Agric. Biol. Chem., 46, 561-565. (8) Matsushita, K., Shinagawa, E., Adachi, O. and Ameyama, M. (1990): Cytochrome al of Acetobacter aceti is a Cytochrome ba Functioning as Ubiquinol Oxidase. Proc. Natl. Acad. Sci. USA., 87, 9863-9867. (9) Matsushita, K., Nagatani, Y., Shinagawa, E., Adachi, O. and Ameyama, M. (1989): Effect of Extracellular pH on the Respiratory Chain and Energetics of Gluconobacter suboxydans. Agric. Biol. Chem., 53, 2895-2902. (10) Ameyama, M. and Kondo, K. (1966): Carbohydrate Metabolism by the Acetic Acid Bacteria. Part V. On the Vitamin Requirements for the Growth. Agric. Biol. Cnem., 30, 203211. (11) Snimao, M., Yamamoto, H., Ninomiya, K., Kato, N., Adachi, O. Ameyama, M. and Sakazawa, C. (1934): Pyrroloquinoline tlinone as an Essential Growtii Factor forQ a Poly(vinylalcohol)-degrading Symbiont, Pseudomonas sp. VM15C. Agric. Biol. Chem., 48, 2873-2876. (12) Bouvet, O. M. M., Pascal, L. and Grimont, P. A. D. (1939): Taxonomic Diversity of the D-Glucose Oxidation Pathway in the Enterobacteriaceae. Intl. J. Syst.Bacteriol., 39, 6167. (13) Biville, F., Turline, E. and Gasser,. (1991): Mutants of Escherichia coli Producing Pyrroloquinoline Quinone. J. Gen. Microbial., in press.
