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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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Crystal structure of extralong MnP compared with short and long MnPs. (a, b) Superimposition of extralong MnP6 from C. subvermispora (PDB entry 4czn; green), long MnP1 from P. chrysosporium (PDB entry 3m5q; light brown) and short MnP4 from P. ostreatus (PDB entry 4bm1; light blue) with the three Mn2+-binding residues as CPK-coloured sticks, Na+ and Fe3+ as orange spheres and the two Ca2+ ions as yellow spheres (see Fig. 3 ▶a for the Mn2+-binding site). (c) Superimposition of the C-tail extension and neighbouring loop in extralong MnP with the same regions of long and short MnPs. (d–f) Surfaces of C. subvermispora extralong MnP (PDB entry 4czo with Mn2+) and the above long and short MnPs, respectively, showing the main (yellow circles) and propionate (white circles) haem-access channels and the long and extralong C-tails in red [the final residue, in light brown, corresponds to Pro365 in (d), Ala357 in (e) and Ser337 in (f)].
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fig1: Crystal structure of extralong MnP compared with short and long MnPs. (a, b) Superimposition of extralong MnP6 from C. subvermispora (PDB entry 4czn; green), long MnP1 from P. chrysosporium (PDB entry 3m5q; light brown) and short MnP4 from P. ostreatus (PDB entry 4bm1; light blue) with the three Mn2+-binding residues as CPK-coloured sticks, Na+ and Fe3+ as orange spheres and the two Ca2+ ions as yellow spheres (see Fig. 3 ▶a for the Mn2+-binding site). (c) Superimposition of the C-tail extension and neighbouring loop in extralong MnP with the same regions of long and short MnPs. (d–f) Surfaces of C. subvermispora extralong MnP (PDB entry 4czo with Mn2+) and the above long and short MnPs, respectively, showing the main (yellow circles) and propionate (white circles) haem-access channels and the long and extralong C-tails in red [the final residue, in light brown, corresponds to Pro365 in (d), Ala357 in (e) and Ser337 in (f)].

Mentions: We have solved the crystal structure of the extralong MnP6 from C. subvermispora after its heterologous expression (Table 1 ▶). The overall folding is similar to that of other fungal peroxidases, with the structure divided into two domains by the haem group (Fig. 1 ▶a, green; Supplementary Fig. S11). A metal-binding site is located near the haem propionates, being occupied by Na+ in the recombinant enzyme (PDB entry 4czn) and coordinating one Mn2+ ion in PDB entry 4czo (from crystals soaked in metal-ion solution). The upper domain is mainly helical with a first structural Ca2+ ion, whereas the lower domain contains α-helices and a non-ordered region stabilized by a second Ca2+ ion. The most significant feature is the extralong tail described below in the C-terminal region (Fig. 1 ▶b). Most of the tail is well defined (Fig. 2 ▶a) and only the last residue (Pro365) that was not visible in the electron-density map and the one before (Ser364) showed significant disorder. The B factors for the residues forming the C-tail (Gly348–Pro365) are comparable to the average for the protein, except for Ser364, which showed a higher value, and the last Pro365, which did not show any electron density in the metal-free form of the protein (Fig. 2 ▶b). The presence of Mn2+ or Cd2+ bound to the protein seems to stabilize the conformation of the last two residues of the C-tail, suggesting that the metal ions might stabilize the enzyme (as described below). A comparison of the experimental B factors of the C-tail residues with those obtained in the MD calculations is shown in Fig. 2 ▶(b). It can be observed that those obtained in the aqueous-medium simulation show higher values than those from the crystal medium. The extra residues that are only present in the extralong forms show a higher mobility in both approaches.


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)

Crystal structure of extralong MnP compared with short and long MnPs. (a, b) Superimposition of extralong MnP6 from C. subvermispora (PDB entry 4czn; green), long MnP1 from P. chrysosporium (PDB entry 3m5q; light brown) and short MnP4 from P. ostreatus (PDB entry 4bm1; light blue) with the three Mn2+-binding residues as CPK-coloured sticks, Na+ and Fe3+ as orange spheres and the two Ca2+ ions as yellow spheres (see Fig. 3 ▶a for the Mn2+-binding site). (c) Superimposition of the C-tail extension and neighbouring loop in extralong MnP with the same regions of long and short MnPs. (d–f) Surfaces of C. subvermispora extralong MnP (PDB entry 4czo with Mn2+) and the above long and short MnPs, respectively, showing the main (yellow circles) and propionate (white circles) haem-access channels and the long and extralong C-tails in red [the final residue, in light brown, corresponds to Pro365 in (d), Ala357 in (e) and Ser337 in (f)].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Crystal structure of extralong MnP compared with short and long MnPs. (a, b) Superimposition of extralong MnP6 from C. subvermispora (PDB entry 4czn; green), long MnP1 from P. chrysosporium (PDB entry 3m5q; light brown) and short MnP4 from P. ostreatus (PDB entry 4bm1; light blue) with the three Mn2+-binding residues as CPK-coloured sticks, Na+ and Fe3+ as orange spheres and the two Ca2+ ions as yellow spheres (see Fig. 3 ▶a for the Mn2+-binding site). (c) Superimposition of the C-tail extension and neighbouring loop in extralong MnP with the same regions of long and short MnPs. (d–f) Surfaces of C. subvermispora extralong MnP (PDB entry 4czo with Mn2+) and the above long and short MnPs, respectively, showing the main (yellow circles) and propionate (white circles) haem-access channels and the long and extralong C-tails in red [the final residue, in light brown, corresponds to Pro365 in (d), Ala357 in (e) and Ser337 in (f)].
Mentions: We have solved the crystal structure of the extralong MnP6 from C. subvermispora after its heterologous expression (Table 1 ▶). The overall folding is similar to that of other fungal peroxidases, with the structure divided into two domains by the haem group (Fig. 1 ▶a, green; Supplementary Fig. S11). A metal-binding site is located near the haem propionates, being occupied by Na+ in the recombinant enzyme (PDB entry 4czn) and coordinating one Mn2+ ion in PDB entry 4czo (from crystals soaked in metal-ion solution). The upper domain is mainly helical with a first structural Ca2+ ion, whereas the lower domain contains α-helices and a non-ordered region stabilized by a second Ca2+ ion. The most significant feature is the extralong tail described below in the C-terminal region (Fig. 1 ▶b). Most of the tail is well defined (Fig. 2 ▶a) and only the last residue (Pro365) that was not visible in the electron-density map and the one before (Ser364) showed significant disorder. The B factors for the residues forming the C-tail (Gly348–Pro365) are comparable to the average for the protein, except for Ser364, which showed a higher value, and the last Pro365, which did not show any electron density in the metal-free form of the protein (Fig. 2 ▶b). The presence of Mn2+ or Cd2+ bound to the protein seems to stabilize the conformation of the last two residues of the C-tail, suggesting that the metal ions might stabilize the enzyme (as described below). A comparison of the experimental B factors of the C-tail residues with those obtained in the MD calculations is shown in Fig. 2 ▶(b). It can be observed that those obtained in the aqueous-medium simulation show higher values than those from the crystal medium. The extra residues that are only present in the extralong forms show a higher mobility in both approaches.

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