JOURNAL OF BACTERIOLOGY, Apr. 1976, p. 516-519 Copyright © 1976 American Society for Microbiology
Vol. 126, No. 1 Printed in U.SA.
NOTES Immunological Properties of Protocatechuate 3,4Dioxygenase Isofunctional Enzymes CHING-TSANG HOU* AND MARJORIE
0.
LILLARD
Corporate Research Laboratories, Exxon Research and Engineering Company, Linden, New Jersey 07036
Received for publication 8 December 1975
Antiserum prepared against protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa forms precipitin zymes from different strains of the
bands without
spurs
with isofunctional
en-
fluorescent pseudomonads on immunodiffusion plates. Catalytic activity of the isofunctional enzymes was inhibited by an immunoglobulin fraction prepared against the enzyme from organisms of the same genus and not from different genera.
Protocatechuate 3,4-dioxygenase (PCD) (protocatechuate:oxygen 3,4-oxidoreductase, EC 1.13.11.3) is a nonheme, trivalent, iron-containing enzyme that catalyzes the transformation of protocatechuic acid to /3-carboxy-cis,cis-muconic acid. The isofunctional dioxygenases have been reported from Pseudomonas aeruginosa (2, 7, 10; C.-T. Hou, R. D. Schwartz, and M. L. Ohaus, Abstr. Annu. Meet. Am. Soc. Microb. 1975, Abstr. RT1, p. 275), Acinetobacter calcoaceticus (C.-T. Hou, M. L. Ohaus, and R. D. Schwartz, Biochemistry, in press), and Thiobacillus (M. Wells, Ph.D. thesis, The University of Texas at Austin, Austin, 1972). They are similar in many aspects, i.e., molecular weight (ca. 700,000), molecular size (19S), subunit size, strict substrate specificity, and iron content (8 gram-atoms/molecule). Recently we isolated and purified PCD from both P. aeruginosa and A. calcoaceticus. In addition to their similarity in general physicochemical properties, the conformation of the active centers of these two isofunctional enzymes is also identical (3; Hou et al., in press). In this paper we report on the immunological properties of PCD isofunctional enzymes. Crystalline PCD from P. aeruginosa and a pure enzyme fraction from A. calcoaceticus were obtained as previously described (Hou et al., in press). Purity and electrophoretic mobility of these two isofunctional enzymes were examined as shown in Fig. 1. Both show a single band on acrylamide gel electropholysis and two distinct bands upon co-electrophoresis. Antiserum against P. aeruginosa PCD was prepared by published procedures (6). The method of Ouchterlony (8) was used for detec-
tion of serological cross-reaction on immunodiffusion plates. The immunoglobulin fraction was obtained by sodium sulfate precipitate (4). All crude PCD fractions were prepared from microorganisms grown with p-hydroxybenzoate as the sole carbon source and inducer. The cells were sonically disintegrated, and the cell-free supernatant fraction (12,000 x g, 30 min) was used as a crude PCD preparation throughout the experiments. One unit of enzyme was defined as the amount that oxidizes 1 ,mol of protocatechuic acid per min at 24 C. Figure 2 shows that an immunoglobulin fraction prepared against P. aeruginosa PCD crossreacts homologously with the isofunctional enzymes from organisms of the genus Pseudomonas and does not cross-react with isofunctional enzymes from other genera. All Pseudomonas strains tested belong to the fluorescent pigment-producing species in section I of the genus Pseudomonas (1). Species in other sections of Pseudomonas were not tested since they produce completely different dioxygenases (i.e., protocatechuate 4,5-dioxygenase) onp-hydroxybenzoate media. The influence of immunoglobulin prepared against P. aeruginosa PCD on the catalytic activity of the isofunctional enzymes is shown in Fig. 3. The conversion of protocatechuic acid to its product was followed with a Beckman spectrophotometer Acta V at 290 nm as previously described (5). The catalytic activity of pure PCD from P. aeruginosa was inhibited by about 80% by the immunoglobulin fraction prepared against the same enzyme. However, this fraction failed completely to inhibit the catalytic activity of pure PCD from A. calcoaceti516
-~~-.
FIG. 1. Acrylamide gel electrophoretic pattern of PCD from different sources. About 15 Mg of the enzyme was applied to each gel slot. Protein migrated to the anode (battom) at a constant voltage of 300 for 2.5 h in 0.05 M tris(hydroxymethyl)aminomethane-glycine buffer, pH 8.5. Arrow indicates front marker. (A) PCD from A. calcoaceticus, specific enzyme activity, 20. (B) PCD from P. aeruginosa specific enzyme activity, 65. (C) co-electrophoresis of the two enzymes. 517
518
NOTES
J. BACTERIOL.
A
B
S.
..
I
0m
FIG. 2. Immunodiffusion patterns ofP. aeruginosa PCD antibody with isofunctional enzymes from different sources. The center wells contained immunoglobulin fraction (0.5 mg) prepared against PCD from P. aeruginosa. Well 1 in each plate contained pure PCD from P. aeruginosa as a control. The other wells contained crude PCD preparations. (A) Wells 2-6 are cell-free extracts from: P. aeruginosa ATCC 23975; P. oleovorans POIR; A. calcoaceticus; Arthrobacter pascens ERC 858; and P. fluorescens ERC 67, respectively. (B) Wells 2-6 contained cell-free extracts from: P. putida; Bacillus subtilis ATCC 6633; pure PCD from A. calcoaceticus; Alcaligenes eutrophus; and Norcadia opaca, respectively. Other cell-free extracts that were tested and showed no precipitation band were from: A. calcoaceticus; Arthrobacter crystallopoietes ATCC 15481; Bacillus stearothermophilous ATIO; Brevibacterium flavium ERC 15168; Brevibacterium fuscum ATCC 15993; Corynebacterium sp. ATCC 21235; Corynebacterium sp. ATCC 21236; Micrococcus cerificans ATCC 14987; and Brevibacterium albus ERC 15111.
loo 1
80
^6 60 40 A
*
c
20._/--°-----......... ______-_s 100
200 IMMUNOGLOBULIN
300
. 400
(,g)
cus. The catalytic activities of the crude PCD preparations from other strains of the genus Pseudomonas were also inhibited by the immunoglobulin fraction prepared against PCD from the same genus. The magnitude of inhibition was somewhat less when crude enzyme preparations were used. The same immunoglobulin shows no inhibition on the catalytic ~.:.~:.~:;fraction cc .. .--~.~~ activities of isofunctional enzymes from Acinetobacter, Nocardia, Arthrobacter, and Thiobacillus. These results indicate that the immunofunctional group(s) of PCD is located in the vicinity of the catalytically active site and is
FIG. 3. Inhibition of PCD catalytic activity by the immunoglobulin fraction from antiserum prepared against PCD from P. aeruginosa. Varying amounts of the immunoglobulin fraction were incubated with isofunctional enzymes (amounts as indicated) in the
different from genus to genus. The serological properties of isofunctional enzymes have been demonstrated (19, 11). Antisera prepared against P. putida muconolactone
enzyme assay mixture (lacking substrate) for 15 min. The enzyme assay was then initiated by adding substrate to the incubation mixture. The uninhibited catalytic activities of each isofunctional PCD in the assay mixture were as follows (in units): pure PCD from (1) P. aeruginosa, 0.15. Cell-free extracts from: (2) P. oleovorans POIR, 0.20; (3) P. aeruginosa
ATCC 23975, 0.36; (4) P. putida, 0.28; and (5) P. fluorescens ERC 67, 0.05. No inhibition was detected (the amount of enzyme used was between 0.1 to 0.3 units) in those cell-free extracts from different microorganisms or pure PCD from A. calcoaceticus, which showed no formation of precipitation band in Fig. 2.
isomerase and muconate lactonizing enzyme do
NOTES
VOL. 126, 1976
not precipitate the isofunctional enzymes from Acinetobacter and Alcaligenes. However, heterologous cross-reactions were observed among the isofunctional enzymes from Pseudomonas. Much heavier spurring was observed with antimuconate lactonizing enzyme than with antimuconolactone isomerase. Our results indicate that the immunoglobulin fractions prepared against P. aeruginosa PCD form a homologous precipitation band and also inhibit the catalytic activity of the isofunctional enzymes from the same genus.
4. 5. 6.
7.
8.
LITERATURE CITED 1. Doudoroff, M., and N. J. Palleroni. 1974. Genus I. Pseudomonas Migula 1894, 237 Nom. cons. Opin. 5, Jud. Comm. 1952, 121, p. 217-243. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey's manual of determinative bacteriology, 8th ed. The Williams & Wilkins
Co., Baltimore. 2. Fujisawa, H., and 0. Hayaishi. 1968. Protocatechuate 3,4-dioxygenase. I. Crystallization and characterization. J. Biol. Chem. 243:2673-2681. 3. Hou, C. T. 1975. Circular dichroism of holo- and apopro-
9.
10. 11.
519
tocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa. Biochemistry 14:3899-3902. Kekwick, R. A. 1940. The serum protein in multiple myelomatosis. Biochem. J. 34:1248-1257. 6 MacDonald, D. L., R. Y. Stanier, and J. L. Ingraham. 1954. The enzymatic formation of f3-carboxymuconic acid. J. Biol. Chem. 210:809-820. Meagher, R. B., and L. N. Ornston. 1973. Relationships among enzymes of the /3-ketoadipate pathway. I. Properties of cis,cis-muconate lactonizing enzyme and muconolactone isomerase from Pseudomonas putida. Biochemistry 12:3523-3530. Ornston, L. N. 1966. The conversion of catechol and protocatechuate to /3-ketoadipate by Pseudomonas putida. J. Biol. Chem. 241:2673-2681. Ouchterlony, 0. 1953. Antigen-antibody reactions in gels; types of reactions in coordinated systems of diffusion. Acta Pathol. Microbiol. Scand. 32:231-240. Patel, R. N., R. B. Meagher, and L. N. Ornston. 1974. Relationships among enzymes of the /8 ketoadipate pathway. IV. Muconolactone isomerase from Acinetobacter calcoaceticus and Pseudomonas putida. J. Biol. Chem. 249:7410-7419. Stanier, R. Y., and J. L. Ingraham. 1954. Protocatechuic acid oxidase. J. Biol. Chem. 210:799-808. Stanier, R. Y., D. Wachter, C. Gasser, and A. C. Wilson. 1970. Comparative immunological studies of two Pseudomonas enzymes. J. Bacteriol. 102:351-360.
Vol. 126, No. 1 Printed in U.SA.
NOTES Immunological Properties of Protocatechuate 3,4Dioxygenase Isofunctional Enzymes CHING-TSANG HOU* AND MARJORIE
0.
LILLARD
Corporate Research Laboratories, Exxon Research and Engineering Company, Linden, New Jersey 07036
Received for publication 8 December 1975
Antiserum prepared against protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa forms precipitin zymes from different strains of the
bands without
spurs
with isofunctional
en-
fluorescent pseudomonads on immunodiffusion plates. Catalytic activity of the isofunctional enzymes was inhibited by an immunoglobulin fraction prepared against the enzyme from organisms of the same genus and not from different genera.
Protocatechuate 3,4-dioxygenase (PCD) (protocatechuate:oxygen 3,4-oxidoreductase, EC 1.13.11.3) is a nonheme, trivalent, iron-containing enzyme that catalyzes the transformation of protocatechuic acid to /3-carboxy-cis,cis-muconic acid. The isofunctional dioxygenases have been reported from Pseudomonas aeruginosa (2, 7, 10; C.-T. Hou, R. D. Schwartz, and M. L. Ohaus, Abstr. Annu. Meet. Am. Soc. Microb. 1975, Abstr. RT1, p. 275), Acinetobacter calcoaceticus (C.-T. Hou, M. L. Ohaus, and R. D. Schwartz, Biochemistry, in press), and Thiobacillus (M. Wells, Ph.D. thesis, The University of Texas at Austin, Austin, 1972). They are similar in many aspects, i.e., molecular weight (ca. 700,000), molecular size (19S), subunit size, strict substrate specificity, and iron content (8 gram-atoms/molecule). Recently we isolated and purified PCD from both P. aeruginosa and A. calcoaceticus. In addition to their similarity in general physicochemical properties, the conformation of the active centers of these two isofunctional enzymes is also identical (3; Hou et al., in press). In this paper we report on the immunological properties of PCD isofunctional enzymes. Crystalline PCD from P. aeruginosa and a pure enzyme fraction from A. calcoaceticus were obtained as previously described (Hou et al., in press). Purity and electrophoretic mobility of these two isofunctional enzymes were examined as shown in Fig. 1. Both show a single band on acrylamide gel electropholysis and two distinct bands upon co-electrophoresis. Antiserum against P. aeruginosa PCD was prepared by published procedures (6). The method of Ouchterlony (8) was used for detec-
tion of serological cross-reaction on immunodiffusion plates. The immunoglobulin fraction was obtained by sodium sulfate precipitate (4). All crude PCD fractions were prepared from microorganisms grown with p-hydroxybenzoate as the sole carbon source and inducer. The cells were sonically disintegrated, and the cell-free supernatant fraction (12,000 x g, 30 min) was used as a crude PCD preparation throughout the experiments. One unit of enzyme was defined as the amount that oxidizes 1 ,mol of protocatechuic acid per min at 24 C. Figure 2 shows that an immunoglobulin fraction prepared against P. aeruginosa PCD crossreacts homologously with the isofunctional enzymes from organisms of the genus Pseudomonas and does not cross-react with isofunctional enzymes from other genera. All Pseudomonas strains tested belong to the fluorescent pigment-producing species in section I of the genus Pseudomonas (1). Species in other sections of Pseudomonas were not tested since they produce completely different dioxygenases (i.e., protocatechuate 4,5-dioxygenase) onp-hydroxybenzoate media. The influence of immunoglobulin prepared against P. aeruginosa PCD on the catalytic activity of the isofunctional enzymes is shown in Fig. 3. The conversion of protocatechuic acid to its product was followed with a Beckman spectrophotometer Acta V at 290 nm as previously described (5). The catalytic activity of pure PCD from P. aeruginosa was inhibited by about 80% by the immunoglobulin fraction prepared against the same enzyme. However, this fraction failed completely to inhibit the catalytic activity of pure PCD from A. calcoaceti516
-~~-.
FIG. 1. Acrylamide gel electrophoretic pattern of PCD from different sources. About 15 Mg of the enzyme was applied to each gel slot. Protein migrated to the anode (battom) at a constant voltage of 300 for 2.5 h in 0.05 M tris(hydroxymethyl)aminomethane-glycine buffer, pH 8.5. Arrow indicates front marker. (A) PCD from A. calcoaceticus, specific enzyme activity, 20. (B) PCD from P. aeruginosa specific enzyme activity, 65. (C) co-electrophoresis of the two enzymes. 517
518
NOTES
J. BACTERIOL.
A
B
S.
..
I
0m
FIG. 2. Immunodiffusion patterns ofP. aeruginosa PCD antibody with isofunctional enzymes from different sources. The center wells contained immunoglobulin fraction (0.5 mg) prepared against PCD from P. aeruginosa. Well 1 in each plate contained pure PCD from P. aeruginosa as a control. The other wells contained crude PCD preparations. (A) Wells 2-6 are cell-free extracts from: P. aeruginosa ATCC 23975; P. oleovorans POIR; A. calcoaceticus; Arthrobacter pascens ERC 858; and P. fluorescens ERC 67, respectively. (B) Wells 2-6 contained cell-free extracts from: P. putida; Bacillus subtilis ATCC 6633; pure PCD from A. calcoaceticus; Alcaligenes eutrophus; and Norcadia opaca, respectively. Other cell-free extracts that were tested and showed no precipitation band were from: A. calcoaceticus; Arthrobacter crystallopoietes ATCC 15481; Bacillus stearothermophilous ATIO; Brevibacterium flavium ERC 15168; Brevibacterium fuscum ATCC 15993; Corynebacterium sp. ATCC 21235; Corynebacterium sp. ATCC 21236; Micrococcus cerificans ATCC 14987; and Brevibacterium albus ERC 15111.
loo 1
80
^6 60 40 A
*
c
20._/--°-----......... ______-_s 100
200 IMMUNOGLOBULIN
300
. 400
(,g)
cus. The catalytic activities of the crude PCD preparations from other strains of the genus Pseudomonas were also inhibited by the immunoglobulin fraction prepared against PCD from the same genus. The magnitude of inhibition was somewhat less when crude enzyme preparations were used. The same immunoglobulin shows no inhibition on the catalytic ~.:.~:.~:;fraction cc .. .--~.~~ activities of isofunctional enzymes from Acinetobacter, Nocardia, Arthrobacter, and Thiobacillus. These results indicate that the immunofunctional group(s) of PCD is located in the vicinity of the catalytically active site and is
FIG. 3. Inhibition of PCD catalytic activity by the immunoglobulin fraction from antiserum prepared against PCD from P. aeruginosa. Varying amounts of the immunoglobulin fraction were incubated with isofunctional enzymes (amounts as indicated) in the
different from genus to genus. The serological properties of isofunctional enzymes have been demonstrated (19, 11). Antisera prepared against P. putida muconolactone
enzyme assay mixture (lacking substrate) for 15 min. The enzyme assay was then initiated by adding substrate to the incubation mixture. The uninhibited catalytic activities of each isofunctional PCD in the assay mixture were as follows (in units): pure PCD from (1) P. aeruginosa, 0.15. Cell-free extracts from: (2) P. oleovorans POIR, 0.20; (3) P. aeruginosa
ATCC 23975, 0.36; (4) P. putida, 0.28; and (5) P. fluorescens ERC 67, 0.05. No inhibition was detected (the amount of enzyme used was between 0.1 to 0.3 units) in those cell-free extracts from different microorganisms or pure PCD from A. calcoaceticus, which showed no formation of precipitation band in Fig. 2.
isomerase and muconate lactonizing enzyme do
NOTES
VOL. 126, 1976
not precipitate the isofunctional enzymes from Acinetobacter and Alcaligenes. However, heterologous cross-reactions were observed among the isofunctional enzymes from Pseudomonas. Much heavier spurring was observed with antimuconate lactonizing enzyme than with antimuconolactone isomerase. Our results indicate that the immunoglobulin fractions prepared against P. aeruginosa PCD form a homologous precipitation band and also inhibit the catalytic activity of the isofunctional enzymes from the same genus.
4. 5. 6.
7.
8.
LITERATURE CITED 1. Doudoroff, M., and N. J. Palleroni. 1974. Genus I. Pseudomonas Migula 1894, 237 Nom. cons. Opin. 5, Jud. Comm. 1952, 121, p. 217-243. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey's manual of determinative bacteriology, 8th ed. The Williams & Wilkins
Co., Baltimore. 2. Fujisawa, H., and 0. Hayaishi. 1968. Protocatechuate 3,4-dioxygenase. I. Crystallization and characterization. J. Biol. Chem. 243:2673-2681. 3. Hou, C. T. 1975. Circular dichroism of holo- and apopro-
9.
10. 11.
519
tocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa. Biochemistry 14:3899-3902. Kekwick, R. A. 1940. The serum protein in multiple myelomatosis. Biochem. J. 34:1248-1257. 6 MacDonald, D. L., R. Y. Stanier, and J. L. Ingraham. 1954. The enzymatic formation of f3-carboxymuconic acid. J. Biol. Chem. 210:809-820. Meagher, R. B., and L. N. Ornston. 1973. Relationships among enzymes of the /3-ketoadipate pathway. I. Properties of cis,cis-muconate lactonizing enzyme and muconolactone isomerase from Pseudomonas putida. Biochemistry 12:3523-3530. Ornston, L. N. 1966. The conversion of catechol and protocatechuate to /3-ketoadipate by Pseudomonas putida. J. Biol. Chem. 241:2673-2681. Ouchterlony, 0. 1953. Antigen-antibody reactions in gels; types of reactions in coordinated systems of diffusion. Acta Pathol. Microbiol. Scand. 32:231-240. Patel, R. N., R. B. Meagher, and L. N. Ornston. 1974. Relationships among enzymes of the /8 ketoadipate pathway. IV. Muconolactone isomerase from Acinetobacter calcoaceticus and Pseudomonas putida. J. Biol. Chem. 249:7410-7419. Stanier, R. Y., and J. L. Ingraham. 1954. Protocatechuic acid oxidase. J. Biol. Chem. 210:799-808. Stanier, R. Y., D. Wachter, C. Gasser, and A. C. Wilson. 1970. Comparative immunological studies of two Pseudomonas enzymes. J. Bacteriol. 102:351-360.
