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Structural implications of the C-terminal tail in the catalytic and stability properties of manganese peroxidases from ligninolytic fungi.

Fernández-Fueyo E, Acebes S, Ruiz-Dueñas FJ, Martínez MJ, Romero A, Medrano FJ, Guallar V, Martínez AT - Acta Crystallogr. D Biol. Crystallogr. (2014)

Bottom Line: The tail, which is anchored by numerous contacts, not only affects the catalytic properties of long/extralong MnPs but is also associated with their high acidic stability.This agrees with molecular simulations that position ABTS at an electron-transfer distance from the haem propionates of an in silico shortened-tail form, while it cannot reach this position in the extralong MnP crystal structure.Only small differences exist between the long and the extralong MnPs, which do not justify their classification as two different subfamilies, but they significantly differ from the short MnPs, with the presence/absence of the C-terminal tail extension being implicated in these differences.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.

ABSTRACT
The genome of Ceriporiopsis subvermispora includes 13 manganese peroxidase (MnP) genes representative of the three subfamilies described in ligninolytic fungi, which share an Mn(2+)-oxidation site and have varying lengths of the C-terminal tail. Short, long and extralong MnPs were heterologously expressed and biochemically characterized, and the first structure of an extralong MnP was solved. Its C-terminal tail surrounds the haem-propionate access channel, contributing to Mn(2+) oxidation by the internal propionate, but prevents the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), which is only oxidized by short MnPs and by shortened-tail variants from site-directed mutagenesis. The tail, which is anchored by numerous contacts, not only affects the catalytic properties of long/extralong MnPs but is also associated with their high acidic stability. Cd(2+) binds at the Mn(2+)-oxidation site and competitively inhibits oxidation of both Mn(2+) and ABTS. Moreover, mutations blocking the haem-propionate channel prevent substrate oxidation. This agrees with molecular simulations that position ABTS at an electron-transfer distance from the haem propionates of an in silico shortened-tail form, while it cannot reach this position in the extralong MnP crystal structure. Only small differences exist between the long and the extralong MnPs, which do not justify their classification as two different subfamilies, but they significantly differ from the short MnPs, with the presence/absence of the C-terminal tail extension being implicated in these differences.

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pH and temperature stabilities. (a) pH stability after incubating the C. subvermispora extralong, long and short MnPs and their short variants (obtained by partial removal of the C-tail) in the pH range 2–8 for 24 h at 4°C (the results at pH 4 and pH 6 were similar to those obtained at pH 5). (b) Temperature stability after incubating the same MnPs and variants in the 25–55°C range for 10 min at pH 5 (the corresponding T50 values are included).
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fig4: pH and temperature stabilities. (a) pH stability after incubating the C. subvermispora extralong, long and short MnPs and their short variants (obtained by partial removal of the C-tail) in the pH range 2–8 for 24 h at 4°C (the results at pH 4 and pH 6 were similar to those obtained at pH 5). (b) Temperature stability after incubating the same MnPs and variants in the 25–55°C range for 10 min at pH 5 (the corresponding T50 values are included).

Mentions: All of the C. subvermispora native MnPs are relatively stable at pH 3, retaining over 75% activity after 24 h (Fig. 4 ▶a). However, strong differences were observed at pH 2, with the long and extralong MnPs maintaining over 60 and 90% activity, respectively, while the short MnP was very quickly (1 min) fully inactivated. Shortening the C-tail affects the pH stability, and the two short variants lost the stability at pH 2 characteristic of the native forms. With regard to temperature, a rough inverse correlation was found between the length of the C-tail and the thermal stability of the native forms (Fig. 4 ▶b), with the short MnP being more thermostable (T50 ≃ 55°C) than the other native MnPs (T50 ≃ 38–45°C). Shortening the C-tails resulted in variants with lower T50 values.


Structural implications of the C-terminal tail in the catalytic and stability properties of manganese peroxidases from ligninolytic fungi.

Fernández-Fueyo E, Acebes S, Ruiz-Dueñas FJ, Martínez MJ, Romero A, Medrano FJ, Guallar V, Martínez AT - Acta Crystallogr. D Biol. Crystallogr. (2014)

pH and temperature stabilities. (a) pH stability after incubating the C. subvermispora extralong, long and short MnPs and their short variants (obtained by partial removal of the C-tail) in the pH range 2–8 for 24 h at 4°C (the results at pH 4 and pH 6 were similar to those obtained at pH 5). (b) Temperature stability after incubating the same MnPs and variants in the 25–55°C range for 10 min at pH 5 (the corresponding T50 values are included).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4257621&req=5

fig4: pH and temperature stabilities. (a) pH stability after incubating the C. subvermispora extralong, long and short MnPs and their short variants (obtained by partial removal of the C-tail) in the pH range 2–8 for 24 h at 4°C (the results at pH 4 and pH 6 were similar to those obtained at pH 5). (b) Temperature stability after incubating the same MnPs and variants in the 25–55°C range for 10 min at pH 5 (the corresponding T50 values are included).
Mentions: All of the C. subvermispora native MnPs are relatively stable at pH 3, retaining over 75% activity after 24 h (Fig. 4 ▶a). However, strong differences were observed at pH 2, with the long and extralong MnPs maintaining over 60 and 90% activity, respectively, while the short MnP was very quickly (1 min) fully inactivated. Shortening the C-tail affects the pH stability, and the two short variants lost the stability at pH 2 characteristic of the native forms. With regard to temperature, a rough inverse correlation was found between the length of the C-tail and the thermal stability of the native forms (Fig. 4 ▶b), with the short MnP being more thermostable (T50 ≃ 55°C) than the other native MnPs (T50 ≃ 38–45°C). Shortening the C-tails resulted in variants with lower T50 values.

Bottom Line: The tail, which is anchored by numerous contacts, not only affects the catalytic properties of long/extralong MnPs but is also associated with their high acidic stability.This agrees with molecular simulations that position ABTS at an electron-transfer distance from the haem propionates of an in silico shortened-tail form, while it cannot reach this position in the extralong MnP crystal structure.Only small differences exist between the long and the extralong MnPs, which do not justify their classification as two different subfamilies, but they significantly differ from the short MnPs, with the presence/absence of the C-terminal tail extension being implicated in these differences.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.

ABSTRACT
The genome of Ceriporiopsis subvermispora includes 13 manganese peroxidase (MnP) genes representative of the three subfamilies described in ligninolytic fungi, which share an Mn(2+)-oxidation site and have varying lengths of the C-terminal tail. Short, long and extralong MnPs were heterologously expressed and biochemically characterized, and the first structure of an extralong MnP was solved. Its C-terminal tail surrounds the haem-propionate access channel, contributing to Mn(2+) oxidation by the internal propionate, but prevents the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), which is only oxidized by short MnPs and by shortened-tail variants from site-directed mutagenesis. The tail, which is anchored by numerous contacts, not only affects the catalytic properties of long/extralong MnPs but is also associated with their high acidic stability. Cd(2+) binds at the Mn(2+)-oxidation site and competitively inhibits oxidation of both Mn(2+) and ABTS. Moreover, mutations blocking the haem-propionate channel prevent substrate oxidation. This agrees with molecular simulations that position ABTS at an electron-transfer distance from the haem propionates of an in silico shortened-tail form, while it cannot reach this position in the extralong MnP crystal structure. Only small differences exist between the long and the extralong MnPs, which do not justify their classification as two different subfamilies, but they significantly differ from the short MnPs, with the presence/absence of the C-terminal tail extension being implicated in these differences.

Show MeSH
Related in: MedlinePlus