/. Blochem. 86, 1159-1162 (1979)
COMMUNICATION
Masayoshi IWAKI, Hiroyuki KAGAMIYAMA, and Mitsuhiro NOZAKI Department of Biochemistry, Shiga University of Medical Science, Ohtsu, Shiga 520-21 Received for publication, July 19, 1979
The complete amino acid sequence of the /9-subunit of protocatechuate 3,4-dioxygenase is presented. The /9-subunit contained 237 amino acid residues, 4 of which were methionines. Accordingly, cyanogen bromide cleavage of the S-carboxymethylated /3-subunit produced five peptides. The sequences of these peptides were determined by analyses of the peptides obtained by tryptic, staphylococcal protease and thermolysin digestions. The alignment of the cyanogen bromide peptides was deduced by the use of overlapping peptides containing methionine which were obtained by tryptic digestion of the S-carboxymethylated /3-subunit. The calculated molecular weight was 26,588, which is close to the value estimated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.
Protocatechuate 3,4-dioxygenase [Protocatechuate: Oxygen 3,4-oxidoreductase, EC 1.13.11.3] catalyzes the intradiol cleavage of the benzene ring of protocatechuic acid with insertion of two atoms of molecular oxygen to form /9-carboxymuconic acid (1). The enzyme (MW 700,000) was obtained in a crystalline form from Pseudomonas aeruginosa (2) and shown to consist of eight identical protomers (MW 90,000), each containing two pairs of two 1
This work was supported in part by a grant from the Naito Foundation and by Grants-in-aid for Scientific Research (144077 and 367066) and for Developmental Scientific Research (387066) from the Ministry of Education, Science and Culture of Japan. The data were taken from a dissertation to be given by M. Iwaki in 1979 to the Graduate School of Kyoto University in partial fulfilment of the requirements of a degree of Doctor of Medical Science. Vol. 86, No. 4, 1979
non-identical subunits, a (MW 22,500) and § (MW 25,000) (3-5). This protomer (atPt) appears to contain one atom of ferric iron, forming one active site of the enzyme (4, 5). Although extensive studies on the reaction mechanism of dioxygenases have been carried out (6-8), the primary structure of none of these enzymes has been established. As the first step toward elucidation and comparison of their primary structures, sequence analysis of protocatechuate 3,4-dioxygenase was carried out. In this paper, the complete amino acid sequence of the /3-subunit is reported. Protocatechuate 3,4-dioxygenase was prepared by the procedure of Fuj'isawa and Hayaishi (2) with some modifications. The preparation of the /3subunit of the enzyme and its 5-carboxymethylation were carried out as described previously (4).
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The Complete Amino Acid Sequence of the /3-Subunit of Protocatechuate 3,4-Dioxygenase from Pseudomonas aeruginosa1
CB-1 40 50 60 Ser-Pro-Arg-Gln-Ala-Leu-Val-Ser-Ile-Pro-Gln-Ser-Ile-Ser-Glu-Thr-Thr-Gly-Pro-Asn-Phe-Ser-His-Leu-Gly-Phe-Gly-Ala-Hls-AspCB-1 70 80 90 Asp-Asp-Leu-Leu-Leu-Asn-Phe-Asn-Asp-Gly-Leu-Pro-Ile-Gly-Glu-Arg-Ile-Ile-Val-Ala-Gly-Arg-Val-Val-Asp-Gln-Tyr-Gly-Lys-ProCB-1
100 110 120 Val-Pro-ABn-Thr-Leu-Val-Glu-Met-Trp-Gln-Ala-Asn-Ala-Gly-Gly-Arg-Tyr-Arg-Hia-Lys-Asn-Asp-Arg-Tyr-Leu-Ala-Pro-Leu-Asp-ProCB-1 1 CB-2 — T-l 130 J.40 150 Asn-Phe-Gly-Gly-Val-Gly-Arg-Cys-Leu-Thr-Asp-Ser-Asp-Gly-Tyr-Tyr-Ser-Phe-Arg-Thr-Ile-Lys-Pro-Gly-Pro-Tyr-Pro-Trp-Arg-AsnCB-2 160 170 180 Gly-Pro-Asn-Asp-Trp-Arg-Pro-Ala-Hls-Ile-His-Phe-Gly-Ile-Ser-Gly'-Pro-Ser-Ile-Ala-Thr-Lys-Leu-Ile-Thr-Gln-Leu-Tyr-Phe-GluCB-2 190 200 210 Gly-Asp-Pro-Leu-Ile-Pro-Met-Cys-Pro-Ile-Val-Lys-Ser-Ile-Ala-Asn-Pro-Glu-Ala-Val-Gln-Gln-Leu-Ile-Ala-Lys-Leu-Asp-Met-AsnCB-2 1 CB-3 1 I X-2 , | 220 230 Asn-Ala-Asn-Pro-Met-Asn-Cys-Leu-Ala-Tyr-Arg-Phe-Asp-Ile-Val-Leu-Arg-Gly-Gln-Arg-Lys-Thr-His-Phe-Glu-Asn-Cys CB-5 1 CB-4 1 T_1
I
|
g_ 3
Fig. I. Primary structure of the /9-subunit of protocatechuate 3,4-dioxygenase. CB-and T-refer to cyanogen bromide peptides and tryptic peptides containing methionine, respectively, obtained from the S-carboxymethylated ^-subunit.
o
| |< S S >
jj 2
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10 20 30 Pro-Ala-Gln-Asp-Asn-Ser-Arg-Phe-Val-Ile-Arg-Asp-Arg-Asn-Trp-His-Pro-Lys-Ala-Leu-Thr-Pro-Asp-Tyr-Lys-Thr-Ser-Ile-Ala-Arg-
PRIMARY STRUCTURE OF THE 0-SUBUNTT OF PROTOCATECHUATE 3,4-DIOXYGENASE
Vol. 86, No. 4, 1979
manual Edman degradation. CB1 was assigned as the NHi-terminal peptide since the NH,-terminal sequence coincided with that reported for the ^-subunit (4). CB4 was assigned as the COOH-terminal peptide since this was the only peptide which did not contain a homoserine residue. The alignment of the cyanogen bromide peptides was established as CB1-CB2CB3-CB5-CB4 by examining the overlapping of the sequences of the 3 methionine-containing peptides obtained by tryptic cleavage of the Scarboxymethylated /9-subunit. One of these peptides contained 2 methionine residues. The sequence studies described above gave the complete amino acid sequence of the 0-subunit as shown in Fig. 1, indicating that the subunit consists of 237 amino acid residues. The amino acid composition of the subunit was Trp4, Lys,, His,, Arg17, Aspn, Asn17, Thr10, Seru, Glu,, Gin,, Proji, Gly19, Alaie, Cys4, Val n , Met4, ne17, Leul8, Tyre, and Phe™. Therefore, the calculated molecular weight was 26,588, which is close to that reported previously (4). However, considering these results, we have to correct some of the results reported previously (4). The subunit contained 4 methionine residues which was reported to be 2 in the previous paper, probably due to the loss of some methionine residues by oxidation during analysis. The amino acid residue at the 6th position was found to be serine which is also inconsistent with the previous paper in which it was reported to be alanine. This discrepancy is probably due to the insufficient differentiation of alanine and serine in the previous experiment. Furthermore, the COOH-terminal amino acid residue which was not determined by three different methods in the previous experiment was now found to be carboxymethylcysteine. The total cysteine residues found in the /3-subunit determined as carboxymethylcysteine were 4. Among them, the possibility of the involvement of disulfide linkages is now under investigation in our laboratory.
The authors would like to express their appreciation to Professor O. Hayaishi of Kyoto University for his continuous encouragement during the course of this investigation. Thanks are also due to Drs. H. Matsubara and T. Hase of Osaka University for their valuable discussions and for making the solid phase sequenator
Downloaded from https://academic.oup.com/jb/article-abstract/86/4/1159/770774 by Stockholm University Library user on 05 January 2019
Trypsin treated with tosylphenylalaninechloromethyl ketone was a product of Worthington, protease from Staphylococcus aureus V-8 strain was a product of Miles, and thermolysin was a gift from Dr. H. Matsubara of Osaka University. Amino acids were analyzed with an automatic amino acid analyzer, JEOL JLC-6AH. Determination of the sequence was carried out either with a liquid phase sequenator, JEOL JAS-47K, a solid phase sequenator, LKB 4020, or by manual Edman degradation as described by BlombSck et al. (9). Identification of phenylthiohydantoin derivatives of amino acids was performed mainly by thin layer chromatography (70). In some cases, free amino acids were identified after conversion of the derivatives to their parent amino acids (11). The peptides obtained by cyanogen bromide treatment (12) of the S-carboxymethylated /Ssubunit were fractionated by Sephadex G-75 chromatography in 30% acetic acid. Purification of the peptides obtained by proteolytic cleavage of the cyanogen bromide peptides was achieved by sequential use of Bio-Gel column chromatography, paper chromatography, and high voltage paper electrophoresis. Isolation of the methionine containing tryptic peptides was carried out by the method of Shechter et al. (13). The cyanogen bromide cleavage of the Scarboxymethylated /3-subunit produced five peptides, which accounted for the entire polypeptide chain. These peptides were designated as CB1 to CB5 in the order of elution from the Sephadex G-75 column chromatography. CB1 contained 98 amino acid residues, and the NH,-terminal sequence up to 30 residues was determined with the solid phase sequenator. The total amino acid sequence of CB1 was established by sequence analyses of the peptides obtained by trypsin, thermolysin, and staphylococcal protease digestions, and by examining the overlapping of their sequences. CB2 contained 89 amino acid residues, and the NH,-terminal sequence was determined up to 21 residues with the liquid phase sequenator. The total sequence of CB2 was established by examining the sequences of tryptic and thermolytic peptides. The sequence of CB3 was established by manual Edman degradation and analyses of the 3 peptides obtained by the tryptic digestion. The sequences of CB4 and CB5 were determined by
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available for us, and to Dr. T. Murachi of Kyoto University for making the liquid phase sequenator available for us.
1. Stanier, R.Y. & Ingraham, J.L. (1954) / . Biol. Chem. 210, 799-808 2. Fujisawa, H. & Hayaishi, O. (1968) / . Biol. Chem. 243, 2673-2681 3. Fujisawa, H., Uyeda, M , Kojima, Y., Nozaki, M., & Hayaishi, O. (1972) / . Biol. Chem. 247, 44144421 4. Yoshida, R., Hori, H., Fujiwara, M., Saeki, Y., Kagamiyama, H., & Nozaki, M. (1976) Biochemistry 15,4048-4053 5. Nozaki, M., Yoshida, R., Nakai, C , Iwaki, M., Saeki, Y., & Kagamiyama, H. (1976) in Iron and Copper Proteins (Yasunobu, K.T., Mower, H.F., & Hayaishi, O., eds.) pp. 127-136, Plenum Publishing Corp., New York
J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/86/4/1159/770774 by Stockholm University Library user on 05 January 2019
REFERENCES
6. Fujisawa, H., Hiromi, K., Uyeda, M., Okuno, S., Nozaki, M., & Hayaishi, O. (1972) / . Biol. Chem. 247, 4422-4428 7. Nozaki, M. (1974) in Molecular Mechanisms of Oxygen Activation (Hayaishi, O., ed.) pp. 135-165, Academic Press, New York 8. Nozaki, M. (1979) in Topics in Current Chemistry Vol. 78 Projected volume "Biochemistry" (Boshke, F.L., ed.) pp. 145-186, Springer-Vcrlag, Heidelberg 9. Blomback, B., Blomback, M., Edman, P., & Hassel, B. (1966) Biochim. Biophys. Acta 115, 371-396 10. Jeppsson, J.O. & SjSquist, J. (1967) Anal. Biochem. 18, 264-269 11. Mendez, E. & Lai, C.Y. (1975) Anal. Biochem. 68, 47-53 12. Steers, E., Jr., Graven, G.R., Anfinsen, C.B., & Bethune, J.L. (1965) / . Biol. Chem. 240, 2478-2484 13. Shechter, Y., Rubinstein, M., & Patchornik, A. (1977) Biochemistry 16, 1424-1430
COMMUNICATION
Masayoshi IWAKI, Hiroyuki KAGAMIYAMA, and Mitsuhiro NOZAKI Department of Biochemistry, Shiga University of Medical Science, Ohtsu, Shiga 520-21 Received for publication, July 19, 1979
The complete amino acid sequence of the /9-subunit of protocatechuate 3,4-dioxygenase is presented. The /9-subunit contained 237 amino acid residues, 4 of which were methionines. Accordingly, cyanogen bromide cleavage of the S-carboxymethylated /3-subunit produced five peptides. The sequences of these peptides were determined by analyses of the peptides obtained by tryptic, staphylococcal protease and thermolysin digestions. The alignment of the cyanogen bromide peptides was deduced by the use of overlapping peptides containing methionine which were obtained by tryptic digestion of the S-carboxymethylated /3-subunit. The calculated molecular weight was 26,588, which is close to the value estimated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.
Protocatechuate 3,4-dioxygenase [Protocatechuate: Oxygen 3,4-oxidoreductase, EC 1.13.11.3] catalyzes the intradiol cleavage of the benzene ring of protocatechuic acid with insertion of two atoms of molecular oxygen to form /9-carboxymuconic acid (1). The enzyme (MW 700,000) was obtained in a crystalline form from Pseudomonas aeruginosa (2) and shown to consist of eight identical protomers (MW 90,000), each containing two pairs of two 1
This work was supported in part by a grant from the Naito Foundation and by Grants-in-aid for Scientific Research (144077 and 367066) and for Developmental Scientific Research (387066) from the Ministry of Education, Science and Culture of Japan. The data were taken from a dissertation to be given by M. Iwaki in 1979 to the Graduate School of Kyoto University in partial fulfilment of the requirements of a degree of Doctor of Medical Science. Vol. 86, No. 4, 1979
non-identical subunits, a (MW 22,500) and § (MW 25,000) (3-5). This protomer (atPt) appears to contain one atom of ferric iron, forming one active site of the enzyme (4, 5). Although extensive studies on the reaction mechanism of dioxygenases have been carried out (6-8), the primary structure of none of these enzymes has been established. As the first step toward elucidation and comparison of their primary structures, sequence analysis of protocatechuate 3,4-dioxygenase was carried out. In this paper, the complete amino acid sequence of the /3-subunit is reported. Protocatechuate 3,4-dioxygenase was prepared by the procedure of Fuj'isawa and Hayaishi (2) with some modifications. The preparation of the /3subunit of the enzyme and its 5-carboxymethylation were carried out as described previously (4).
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The Complete Amino Acid Sequence of the /3-Subunit of Protocatechuate 3,4-Dioxygenase from Pseudomonas aeruginosa1
CB-1 40 50 60 Ser-Pro-Arg-Gln-Ala-Leu-Val-Ser-Ile-Pro-Gln-Ser-Ile-Ser-Glu-Thr-Thr-Gly-Pro-Asn-Phe-Ser-His-Leu-Gly-Phe-Gly-Ala-Hls-AspCB-1 70 80 90 Asp-Asp-Leu-Leu-Leu-Asn-Phe-Asn-Asp-Gly-Leu-Pro-Ile-Gly-Glu-Arg-Ile-Ile-Val-Ala-Gly-Arg-Val-Val-Asp-Gln-Tyr-Gly-Lys-ProCB-1
100 110 120 Val-Pro-ABn-Thr-Leu-Val-Glu-Met-Trp-Gln-Ala-Asn-Ala-Gly-Gly-Arg-Tyr-Arg-Hia-Lys-Asn-Asp-Arg-Tyr-Leu-Ala-Pro-Leu-Asp-ProCB-1 1 CB-2 — T-l 130 J.40 150 Asn-Phe-Gly-Gly-Val-Gly-Arg-Cys-Leu-Thr-Asp-Ser-Asp-Gly-Tyr-Tyr-Ser-Phe-Arg-Thr-Ile-Lys-Pro-Gly-Pro-Tyr-Pro-Trp-Arg-AsnCB-2 160 170 180 Gly-Pro-Asn-Asp-Trp-Arg-Pro-Ala-Hls-Ile-His-Phe-Gly-Ile-Ser-Gly'-Pro-Ser-Ile-Ala-Thr-Lys-Leu-Ile-Thr-Gln-Leu-Tyr-Phe-GluCB-2 190 200 210 Gly-Asp-Pro-Leu-Ile-Pro-Met-Cys-Pro-Ile-Val-Lys-Ser-Ile-Ala-Asn-Pro-Glu-Ala-Val-Gln-Gln-Leu-Ile-Ala-Lys-Leu-Asp-Met-AsnCB-2 1 CB-3 1 I X-2 , | 220 230 Asn-Ala-Asn-Pro-Met-Asn-Cys-Leu-Ala-Tyr-Arg-Phe-Asp-Ile-Val-Leu-Arg-Gly-Gln-Arg-Lys-Thr-His-Phe-Glu-Asn-Cys CB-5 1 CB-4 1 T_1
I
|
g_ 3
Fig. I. Primary structure of the /9-subunit of protocatechuate 3,4-dioxygenase. CB-and T-refer to cyanogen bromide peptides and tryptic peptides containing methionine, respectively, obtained from the S-carboxymethylated ^-subunit.
o
| |< S S >
jj 2
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10 20 30 Pro-Ala-Gln-Asp-Asn-Ser-Arg-Phe-Val-Ile-Arg-Asp-Arg-Asn-Trp-His-Pro-Lys-Ala-Leu-Thr-Pro-Asp-Tyr-Lys-Thr-Ser-Ile-Ala-Arg-
PRIMARY STRUCTURE OF THE 0-SUBUNTT OF PROTOCATECHUATE 3,4-DIOXYGENASE
Vol. 86, No. 4, 1979
manual Edman degradation. CB1 was assigned as the NHi-terminal peptide since the NH,-terminal sequence coincided with that reported for the ^-subunit (4). CB4 was assigned as the COOH-terminal peptide since this was the only peptide which did not contain a homoserine residue. The alignment of the cyanogen bromide peptides was established as CB1-CB2CB3-CB5-CB4 by examining the overlapping of the sequences of the 3 methionine-containing peptides obtained by tryptic cleavage of the Scarboxymethylated /9-subunit. One of these peptides contained 2 methionine residues. The sequence studies described above gave the complete amino acid sequence of the 0-subunit as shown in Fig. 1, indicating that the subunit consists of 237 amino acid residues. The amino acid composition of the subunit was Trp4, Lys,, His,, Arg17, Aspn, Asn17, Thr10, Seru, Glu,, Gin,, Proji, Gly19, Alaie, Cys4, Val n , Met4, ne17, Leul8, Tyre, and Phe™. Therefore, the calculated molecular weight was 26,588, which is close to that reported previously (4). However, considering these results, we have to correct some of the results reported previously (4). The subunit contained 4 methionine residues which was reported to be 2 in the previous paper, probably due to the loss of some methionine residues by oxidation during analysis. The amino acid residue at the 6th position was found to be serine which is also inconsistent with the previous paper in which it was reported to be alanine. This discrepancy is probably due to the insufficient differentiation of alanine and serine in the previous experiment. Furthermore, the COOH-terminal amino acid residue which was not determined by three different methods in the previous experiment was now found to be carboxymethylcysteine. The total cysteine residues found in the /3-subunit determined as carboxymethylcysteine were 4. Among them, the possibility of the involvement of disulfide linkages is now under investigation in our laboratory.
The authors would like to express their appreciation to Professor O. Hayaishi of Kyoto University for his continuous encouragement during the course of this investigation. Thanks are also due to Drs. H. Matsubara and T. Hase of Osaka University for their valuable discussions and for making the solid phase sequenator
Downloaded from https://academic.oup.com/jb/article-abstract/86/4/1159/770774 by Stockholm University Library user on 05 January 2019
Trypsin treated with tosylphenylalaninechloromethyl ketone was a product of Worthington, protease from Staphylococcus aureus V-8 strain was a product of Miles, and thermolysin was a gift from Dr. H. Matsubara of Osaka University. Amino acids were analyzed with an automatic amino acid analyzer, JEOL JLC-6AH. Determination of the sequence was carried out either with a liquid phase sequenator, JEOL JAS-47K, a solid phase sequenator, LKB 4020, or by manual Edman degradation as described by BlombSck et al. (9). Identification of phenylthiohydantoin derivatives of amino acids was performed mainly by thin layer chromatography (70). In some cases, free amino acids were identified after conversion of the derivatives to their parent amino acids (11). The peptides obtained by cyanogen bromide treatment (12) of the S-carboxymethylated /Ssubunit were fractionated by Sephadex G-75 chromatography in 30% acetic acid. Purification of the peptides obtained by proteolytic cleavage of the cyanogen bromide peptides was achieved by sequential use of Bio-Gel column chromatography, paper chromatography, and high voltage paper electrophoresis. Isolation of the methionine containing tryptic peptides was carried out by the method of Shechter et al. (13). The cyanogen bromide cleavage of the Scarboxymethylated /3-subunit produced five peptides, which accounted for the entire polypeptide chain. These peptides were designated as CB1 to CB5 in the order of elution from the Sephadex G-75 column chromatography. CB1 contained 98 amino acid residues, and the NH,-terminal sequence up to 30 residues was determined with the solid phase sequenator. The total amino acid sequence of CB1 was established by sequence analyses of the peptides obtained by trypsin, thermolysin, and staphylococcal protease digestions, and by examining the overlapping of their sequences. CB2 contained 89 amino acid residues, and the NH,-terminal sequence was determined up to 21 residues with the liquid phase sequenator. The total sequence of CB2 was established by examining the sequences of tryptic and thermolytic peptides. The sequence of CB3 was established by manual Edman degradation and analyses of the 3 peptides obtained by the tryptic digestion. The sequences of CB4 and CB5 were determined by
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available for us, and to Dr. T. Murachi of Kyoto University for making the liquid phase sequenator available for us.
1. Stanier, R.Y. & Ingraham, J.L. (1954) / . Biol. Chem. 210, 799-808 2. Fujisawa, H. & Hayaishi, O. (1968) / . Biol. Chem. 243, 2673-2681 3. Fujisawa, H., Uyeda, M , Kojima, Y., Nozaki, M., & Hayaishi, O. (1972) / . Biol. Chem. 247, 44144421 4. Yoshida, R., Hori, H., Fujiwara, M., Saeki, Y., Kagamiyama, H., & Nozaki, M. (1976) Biochemistry 15,4048-4053 5. Nozaki, M., Yoshida, R., Nakai, C , Iwaki, M., Saeki, Y., & Kagamiyama, H. (1976) in Iron and Copper Proteins (Yasunobu, K.T., Mower, H.F., & Hayaishi, O., eds.) pp. 127-136, Plenum Publishing Corp., New York
J. Biochem.
Downloaded from https://academic.oup.com/jb/article-abstract/86/4/1159/770774 by Stockholm University Library user on 05 January 2019
REFERENCES
6. Fujisawa, H., Hiromi, K., Uyeda, M., Okuno, S., Nozaki, M., & Hayaishi, O. (1972) / . Biol. Chem. 247, 4422-4428 7. Nozaki, M. (1974) in Molecular Mechanisms of Oxygen Activation (Hayaishi, O., ed.) pp. 135-165, Academic Press, New York 8. Nozaki, M. (1979) in Topics in Current Chemistry Vol. 78 Projected volume "Biochemistry" (Boshke, F.L., ed.) pp. 145-186, Springer-Vcrlag, Heidelberg 9. Blomback, B., Blomback, M., Edman, P., & Hassel, B. (1966) Biochim. Biophys. Acta 115, 371-396 10. Jeppsson, J.O. & SjSquist, J. (1967) Anal. Biochem. 18, 264-269 11. Mendez, E. & Lai, C.Y. (1975) Anal. Biochem. 68, 47-53 12. Steers, E., Jr., Graven, G.R., Anfinsen, C.B., & Bethune, J.L. (1965) / . Biol. Chem. 240, 2478-2484 13. Shechter, Y., Rubinstein, M., & Patchornik, A. (1977) Biochemistry 16, 1424-1430
