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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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ABTS diffusion by PELE in the vicinity of the haem propionates. (a, b) Interaction energy versus distance between the haem CHA and the ABTS mass centre (a) and between protonated Gluh35 CG and Asph179 CD (b) during substrate diffusion at the haem-propionate region of C. subvermispora extralong MnP6 (blue) and its in silico shortened-tail form (red), with the haem–ABTS electron-coupling constants at the most favourable positions indicated (circles). Diffusion simulations were performed with PELE (Borrelli et al., 2005 ▶), fixing a 20 Å cutoff distance between the haem CHA atom and the ABTS centre of mass. (c, d) Detail of the ABTS location and the positions of Glu35, Glu39 and Asp179 in two snapshots from the above substrate diffusion on the extralong MnP6 (c) and the shortened-tail form (d). Residues are shown as CPK-coloured sticks, with ABTS also shown with semitransparent van der Waals spheres. Distances between Dh179 and Eh35, and between ABTS and the most internal propionate of heme are indicated.
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fig8: ABTS diffusion by PELE in the vicinity of the haem propionates. (a, b) Interaction energy versus distance between the haem CHA and the ABTS mass centre (a) and between protonated Gluh35 CG and Asph179 CD (b) during substrate diffusion at the haem-propionate region of C. subvermispora extralong MnP6 (blue) and its in silico shortened-tail form (red), with the haem–ABTS electron-coupling constants at the most favourable positions indicated (circles). Diffusion simulations were performed with PELE (Borrelli et al., 2005 ▶), fixing a 20 Å cutoff distance between the haem CHA atom and the ABTS centre of mass. (c, d) Detail of the ABTS location and the positions of Glu35, Glu39 and Asp179 in two snapshots from the above substrate diffusion on the extralong MnP6 (c) and the shortened-tail form (d). Residues are shown as CPK-coloured sticks, with ABTS also shown with semitransparent van der Waals spheres. Distances between Dh179 and Eh35, and between ABTS and the most internal propionate of heme are indicated.

Mentions: A first free exploration of ABTS diffusion on the whole enzyme was performed with PELE (Supplementary Figs. S4a and S4b). The main energy minimum was identified at the entrance to the haem-propionate channel (site I), where more detailed local explorations were performed (Fig. 8 ▶a). For the shortened-tail form (red dots), the structures with the best interaction energy have ABTS closer to the active site. In contrast, ABTS diffusion on extralong MnP (blue dots) failed to identify a structure with good interaction energy, and ABTS always occupied much more distant positions. Using QM/MM techniques, the electronic coupling was calculated at the best positions predicted, being one order of magnitude higher for the shortened-tail MnP than for the extralong MnP (Fig. 8 ▶a, red and blue text, respectively).


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)

ABTS diffusion by PELE in the vicinity of the haem propionates. (a, b) Interaction energy versus distance between the haem CHA and the ABTS mass centre (a) and between protonated Gluh35 CG and Asph179 CD (b) during substrate diffusion at the haem-propionate region of C. subvermispora extralong MnP6 (blue) and its in silico shortened-tail form (red), with the haem–ABTS electron-coupling constants at the most favourable positions indicated (circles). Diffusion simulations were performed with PELE (Borrelli et al., 2005 ▶), fixing a 20 Å cutoff distance between the haem CHA atom and the ABTS centre of mass. (c, d) Detail of the ABTS location and the positions of Glu35, Glu39 and Asp179 in two snapshots from the above substrate diffusion on the extralong MnP6 (c) and the shortened-tail form (d). Residues are shown as CPK-coloured sticks, with ABTS also shown with semitransparent van der Waals spheres. Distances between Dh179 and Eh35, and between ABTS and the most internal propionate of heme are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig8: ABTS diffusion by PELE in the vicinity of the haem propionates. (a, b) Interaction energy versus distance between the haem CHA and the ABTS mass centre (a) and between protonated Gluh35 CG and Asph179 CD (b) during substrate diffusion at the haem-propionate region of C. subvermispora extralong MnP6 (blue) and its in silico shortened-tail form (red), with the haem–ABTS electron-coupling constants at the most favourable positions indicated (circles). Diffusion simulations were performed with PELE (Borrelli et al., 2005 ▶), fixing a 20 Å cutoff distance between the haem CHA atom and the ABTS centre of mass. (c, d) Detail of the ABTS location and the positions of Glu35, Glu39 and Asp179 in two snapshots from the above substrate diffusion on the extralong MnP6 (c) and the shortened-tail form (d). Residues are shown as CPK-coloured sticks, with ABTS also shown with semitransparent van der Waals spheres. Distances between Dh179 and Eh35, and between ABTS and the most internal propionate of heme are indicated.
Mentions: A first free exploration of ABTS diffusion on the whole enzyme was performed with PELE (Supplementary Figs. S4a and S4b). The main energy minimum was identified at the entrance to the haem-propionate channel (site I), where more detailed local explorations were performed (Fig. 8 ▶a). For the shortened-tail form (red dots), the structures with the best interaction energy have ABTS closer to the active site. In contrast, ABTS diffusion on extralong MnP (blue dots) failed to identify a structure with good interaction energy, and ABTS always occupied much more distant positions. Using QM/MM techniques, the electronic coupling was calculated at the best positions predicted, being one order of magnitude higher for the shortened-tail MnP than for the extralong MnP (Fig. 8 ▶a, red and blue text, respectively).

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