Vol. 129, No. 2 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Feb. 1977, p. 690-697 Copyright © 1977 American Society for Microbiology
Purification and Properties of Homoprotocatechuate 2,3Dioxygenase from Bacillus stearothermophilus' MOIDEEN P. JAMALUDDIN2 Department of Biochemistry, College of Biological Sciences, University of Minnesota, St. Paul, Minnesota 55108 Received for publication 16 August 1976
The enzyme 3,4-dihydroxyphenylacetate:oxygen 2,3-oxidoreductase (decyclizing) (homoprotocatechuate 2,3-dioxygenase) was purified from the thermophilic organism Bacillus stearothermophilus, grown with 4-hydroxyphenylacetic acid as a source of carbon. The enzyme appeared to be homogeneous as judged by disc-gel electrophoresis and sedimentation equilibrium measurements. The average molecular weight determined by three independent procedures was 106,000; the protein was globular and was dissociated in sodium dodecyl sulfate to give a species of molecular weight 33,000 to 35,000. The enzyme was fairly stable on heating and showed maximal activity at about 57°C. An Arrhenius plot of Km for homoprotocatechuate was concave upward, with a break at 32°C; an increase in AHIt above this temperature was compensated by lower values of -ASt. Several properties of this enzyme are contrasted with those reported for homoprotocatechuate 2,3-dioxygenase purified by other workers from Pseudomonas ovalis.
Homoprotocatechuate 2,3-dioxygenase (3,4dihydroxyphenylacetate: oxygen 2, 3 - oxidoreductase [decyclizing]) catalyzes fission of the benzene nucleus of homoprotocatechuate to give 5-carboxymethyl -2 -hydroxy-cis, cis -muconic semialdehyde (Fig. 1). This enzyme, crystallized from Pseudomonas ovalis (9, 17), was described previously (15), and has been assigned the number EC 1.13.11.15. Evidence for fission between C2 and C3 of the benzene nucleus was presented by Adachi et al. (1) and Blakley et al. (2) and is in accordance with the metabolic studies of Sparnins et al. (18). It may be mentioned that the Committee on Biochemical Nomenclature (3) has also assigned the number EC 1.13.11.7 to a homoprotocatechuate oxygenase that is shown as catalyzing fission between C3 and C4. However, the sole reference given is to Kita et al. (10), who studied homoprotocatechuate 2,3-dioxygenase (EC 1.13.11.15), and it therefore appears that the entry EC 1.13.11.7 is redundant. The present paper describes the purification of homoprotocatechuate 2,3-dioxygenase (HP dioxygenase) from the thermophilic microorganism Bacillus stearothermophilus. The properties of the en-
zyme from this source were found to differ in several respects from those reported for the enzyme isolated from Pseudomonas. MATERIALS AND METHODS Isolation of the organism and conditions of culture. The bacillus was isolated by Mark Elstad from grass cuttings fermenting at 65°C. Pasteurization at 80°C for 10 min was followed by selective enrichment for growth with phenylacetic acid at 65°C. The organism was identified as B. stearothermophilus (7). Stock cultures were maintained on nutrient agar (Difco) slants, stored at 4°C, and subcultured at intervals of 2 weeks. The growth medium, adjusted to pH 7 with NaOH, contained (per liter): K2HPO4 * 3H20, 4.25 g; NaH2PO4 H20, 1.0 g; NH4Cl, 2.0 g; MgSO4- 7H20, 0.2 g; FeSO4 7H20, 0.12 g; MnSO4 H20, 0.03 g; ZnSO4 7H2O, 0.003 g; CoSO4, 0.001 g; vitamin-free Casamino Acids (Difco), 0.05 g; yeast extract (Difco), 0.05 g; and 4-hydroxphenylacetic acid, 0.5 g. A stationary culture (50 ml) was grown at 65°C and used to inoculate 1 liter of medium at 60°C in a shaken 2-liter Erlenmeyer flask,
H OOC CH2
HOOC CH2
-
02
' Address reprint requests to: Stanley Dagley, Department of Biochemistry, University of Minnesota, St. Paul, MN 55108. 2 Present address: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
OH
1'FCHO
L
JOURNAL OF BACTERIOLOGY, Feb. 1977, p. 690-697 Copyright © 1977 American Society for Microbiology
Purification and Properties of Homoprotocatechuate 2,3Dioxygenase from Bacillus stearothermophilus' MOIDEEN P. JAMALUDDIN2 Department of Biochemistry, College of Biological Sciences, University of Minnesota, St. Paul, Minnesota 55108 Received for publication 16 August 1976
The enzyme 3,4-dihydroxyphenylacetate:oxygen 2,3-oxidoreductase (decyclizing) (homoprotocatechuate 2,3-dioxygenase) was purified from the thermophilic organism Bacillus stearothermophilus, grown with 4-hydroxyphenylacetic acid as a source of carbon. The enzyme appeared to be homogeneous as judged by disc-gel electrophoresis and sedimentation equilibrium measurements. The average molecular weight determined by three independent procedures was 106,000; the protein was globular and was dissociated in sodium dodecyl sulfate to give a species of molecular weight 33,000 to 35,000. The enzyme was fairly stable on heating and showed maximal activity at about 57°C. An Arrhenius plot of Km for homoprotocatechuate was concave upward, with a break at 32°C; an increase in AHIt above this temperature was compensated by lower values of -ASt. Several properties of this enzyme are contrasted with those reported for homoprotocatechuate 2,3-dioxygenase purified by other workers from Pseudomonas ovalis.
Homoprotocatechuate 2,3-dioxygenase (3,4dihydroxyphenylacetate: oxygen 2, 3 - oxidoreductase [decyclizing]) catalyzes fission of the benzene nucleus of homoprotocatechuate to give 5-carboxymethyl -2 -hydroxy-cis, cis -muconic semialdehyde (Fig. 1). This enzyme, crystallized from Pseudomonas ovalis (9, 17), was described previously (15), and has been assigned the number EC 1.13.11.15. Evidence for fission between C2 and C3 of the benzene nucleus was presented by Adachi et al. (1) and Blakley et al. (2) and is in accordance with the metabolic studies of Sparnins et al. (18). It may be mentioned that the Committee on Biochemical Nomenclature (3) has also assigned the number EC 1.13.11.7 to a homoprotocatechuate oxygenase that is shown as catalyzing fission between C3 and C4. However, the sole reference given is to Kita et al. (10), who studied homoprotocatechuate 2,3-dioxygenase (EC 1.13.11.15), and it therefore appears that the entry EC 1.13.11.7 is redundant. The present paper describes the purification of homoprotocatechuate 2,3-dioxygenase (HP dioxygenase) from the thermophilic microorganism Bacillus stearothermophilus. The properties of the en-
zyme from this source were found to differ in several respects from those reported for the enzyme isolated from Pseudomonas. MATERIALS AND METHODS Isolation of the organism and conditions of culture. The bacillus was isolated by Mark Elstad from grass cuttings fermenting at 65°C. Pasteurization at 80°C for 10 min was followed by selective enrichment for growth with phenylacetic acid at 65°C. The organism was identified as B. stearothermophilus (7). Stock cultures were maintained on nutrient agar (Difco) slants, stored at 4°C, and subcultured at intervals of 2 weeks. The growth medium, adjusted to pH 7 with NaOH, contained (per liter): K2HPO4 * 3H20, 4.25 g; NaH2PO4 H20, 1.0 g; NH4Cl, 2.0 g; MgSO4- 7H20, 0.2 g; FeSO4 7H20, 0.12 g; MnSO4 H20, 0.03 g; ZnSO4 7H2O, 0.003 g; CoSO4, 0.001 g; vitamin-free Casamino Acids (Difco), 0.05 g; yeast extract (Difco), 0.05 g; and 4-hydroxphenylacetic acid, 0.5 g. A stationary culture (50 ml) was grown at 65°C and used to inoculate 1 liter of medium at 60°C in a shaken 2-liter Erlenmeyer flask,
H OOC CH2
HOOC CH2
-
02
' Address reprint requests to: Stanley Dagley, Department of Biochemistry, University of Minnesota, St. Paul, MN 55108. 2 Present address: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
OH
1'FCHO
L
