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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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Cation binding on extralong MnP. (a, b) Anomalous difference electron-density maps showing two Mn2+ (a) and three Cd2+ (b) ions in the proximity of haem (and six acidic residues) in the crystal structures of metal complexes of extralong MnP6 from C. subvermispora (PDB entries 4czp and 4czr, respectively).
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fig6: Cation binding on extralong MnP. (a, b) Anomalous difference electron-density maps showing two Mn2+ (a) and three Cd2+ (b) ions in the proximity of haem (and six acidic residues) in the crystal structures of metal complexes of extralong MnP6 from C. subvermispora (PDB entries 4czp and 4czr, respectively).

Mentions: Binding of Mn2+ and Cd2+ (with only the first being a MnP substrate) was confirmed by anomalous difference electron-density maps of the MnP6–Mn2+ and MnP6–Cd2+ complexes. The first map (Fig. 6 ▶a) showed two Mn2+ ions, one at the predicted Mn-oxidation site, coordinated by Glu35, Glu39, Asp179, the internal (with respect to the main haem-access channel) propionate of haem and two (not shown) water molecules, and the second in a neighbouring position at 6.7 Å from the external propionate. Cd2+ binding was observed at the same two locations, plus a third position at 10.7 Å from the external propionate (Fig. 6 ▶b).


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)

Cation binding on extralong MnP. (a, b) Anomalous difference electron-density maps showing two Mn2+ (a) and three Cd2+ (b) ions in the proximity of haem (and six acidic residues) in the crystal structures of metal complexes of extralong MnP6 from C. subvermispora (PDB entries 4czp and 4czr, respectively).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: Cation binding on extralong MnP. (a, b) Anomalous difference electron-density maps showing two Mn2+ (a) and three Cd2+ (b) ions in the proximity of haem (and six acidic residues) in the crystal structures of metal complexes of extralong MnP6 from C. subvermispora (PDB entries 4czp and 4czr, respectively).
Mentions: Binding of Mn2+ and Cd2+ (with only the first being a MnP substrate) was confirmed by anomalous difference electron-density maps of the MnP6–Mn2+ and MnP6–Cd2+ complexes. The first map (Fig. 6 ▶a) showed two Mn2+ ions, one at the predicted Mn-oxidation site, coordinated by Glu35, Glu39, Asp179, the internal (with respect to the main haem-access channel) propionate of haem and two (not shown) water molecules, and the second in a neighbouring position at 6.7 Å from the external propionate. Cd2+ binding was observed at the same two locations, plus a third position at 10.7 Å from the external propionate (Fig. 6 ▶b).

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