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Inter-conversion of catalytic abilities in a bifunctional carboxyl/feruloyl-esterase from earthworm gut metagenome.

Vieites JM, Ghazi A, Beloqui A, Polaina J, Andreu JM, Golyshina OV, Nechitaylo TY, Waliczek A, Yakimov MM, Golyshin PN, Ferrer M - Microb Biotechnol (2009)

Bottom Line: Although, single to triple mutants with both improved activities (up to 180-fold in k(cat)/K(m) values) and enzymes with inverted specificity ((k(cat)/K(m))(CE)/(k(cat)/K(m))(FAE) ratio of ∼0.4) were identified, no CE inactive variant was found.Screening of a large error-prone PCR-generated library yielded by far less mutants for substrate discrimination.We also found that no significant changes in CE activation energy occurs after any mutation (7.3 to -5.6 J mol(-1)), whereas a direct correlation between loss/gain of FAE function and activation energies (from 33.05 to -13.7 J mol(-1)) was found.

View Article: PubMed Central - PubMed

Affiliation: CSIC, Institute of Catalysis, 28049 Madrid, Spain. CSIC, Instituto de Agroquímica y Tecnología de Alimentos, 46980 Valencia, Spain.

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Surface representation of the substrate access pathways in the 3A6 protein. The upper panel (A, C, E, G) corresponds to the wild‐type protein whereas the bottom panel (B, D, F, H) correspond to the model containing the corresponding mutation. Panels G and H represent the wild‐type protein oriented with a difference of 90°C (in red is shown the C‐terminal part which is removed after N316STOP mutation). In all cases, the catalytic core is shown in green colour, where as the original or new introduced mutation are shown in pink or red colour. Panel I illustrates the view of the Gly178‐Gly211 insertion related to the catalytic core (green).
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f4: Surface representation of the substrate access pathways in the 3A6 protein. The upper panel (A, C, E, G) corresponds to the wild‐type protein whereas the bottom panel (B, D, F, H) correspond to the model containing the corresponding mutation. Panels G and H represent the wild‐type protein oriented with a difference of 90°C (in red is shown the C‐terminal part which is removed after N316STOP mutation). In all cases, the catalytic core is shown in green colour, where as the original or new introduced mutation are shown in pink or red colour. Panel I illustrates the view of the Gly178‐Gly211 insertion related to the catalytic core (green).

Mentions: Since K281, D282, N316 and K317 are situated at specific loci within the catalytic core, their effect in promoting substrate promiscuity can be analysed in combination with the 3D model. K281I exerts a perturbation in the final access tunnel to the catalytic centre, generating a high (34‐fold) or mild (1.4‐fold) improvement in catalytic efficiency for FAE‐ and CE‐like substrates respectively (Fig. 4A and B). Saturation mutagenesis at the position 281 confirmed Ile as the best possible amino acid substitution for improving FAE phenotype. The Ile is a smaller residue making more accessible substrate channel that may correlate with higher values of kcat, whereas the substitution of a hydrophilic (Lys) by a hydrophobic (Ile) residue may correlate with enhanced binding of the substrates (lower Km values). This observation can also be extrapolated to the variant D282L (hydrophilic→hydrophobic); however, here, the slightly larger size of Asp than that of Leu appears to have more impact towards smaller substrates such as pNPC2 (Fig. 4C and D). Lys317His appear to have a significant negative impact on the properties of the enzyme since the mutation was associated with a complete loss of FAE activity. The effect on pNP esters was mostly associated with a twofold decrease in substrate affinity while slightly affecting the reaction rates (kcat). Since it was suggested that the presence of at least one m/p‐methoxy group in the cinnamate‐like substrates is required for binding (Tarbouriech et al., 2005), one could argue that the mutation at His317 may cause a partial unwinding of the loop and shortening the distance between binding residues and cinnamate‐like substrates while maintaining the activity with common substrates (Fig. 4E and F). The mutation Asn316STOP produced a deletion of the C‐terminal tail (25 amino acids) (Fig. 4G and H). This fragment constitutes an α‐helix located back to the catalytic cavity. Although, the deletion does not seem to be directly relevant to the catalytic core, we suggest that it may confer a higher flexibility, making catalysis more efficient, in particular, with shorter substrates. To further assess whether the higher flexibility is produced at expenses of lower structural stability, as suggested by the model, we studied the biophysical parameters of this protein variant after pre‐incubation with different concentrations of guanidinium chloride (GdmCl) and at different temperatures. We showed that this variant tends to misfold to a large degree after pre‐incubation with ≥ 0.64 M GdmCl whereas the wild‐type protein was mostly chemically stable (misfolding occurred ≥ 2.1 M GdmCl) (Fig. S7).


Inter-conversion of catalytic abilities in a bifunctional carboxyl/feruloyl-esterase from earthworm gut metagenome.

Vieites JM, Ghazi A, Beloqui A, Polaina J, Andreu JM, Golyshina OV, Nechitaylo TY, Waliczek A, Yakimov MM, Golyshin PN, Ferrer M - Microb Biotechnol (2009)

Surface representation of the substrate access pathways in the 3A6 protein. The upper panel (A, C, E, G) corresponds to the wild‐type protein whereas the bottom panel (B, D, F, H) correspond to the model containing the corresponding mutation. Panels G and H represent the wild‐type protein oriented with a difference of 90°C (in red is shown the C‐terminal part which is removed after N316STOP mutation). In all cases, the catalytic core is shown in green colour, where as the original or new introduced mutation are shown in pink or red colour. Panel I illustrates the view of the Gly178‐Gly211 insertion related to the catalytic core (green).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815946&req=5

f4: Surface representation of the substrate access pathways in the 3A6 protein. The upper panel (A, C, E, G) corresponds to the wild‐type protein whereas the bottom panel (B, D, F, H) correspond to the model containing the corresponding mutation. Panels G and H represent the wild‐type protein oriented with a difference of 90°C (in red is shown the C‐terminal part which is removed after N316STOP mutation). In all cases, the catalytic core is shown in green colour, where as the original or new introduced mutation are shown in pink or red colour. Panel I illustrates the view of the Gly178‐Gly211 insertion related to the catalytic core (green).
Mentions: Since K281, D282, N316 and K317 are situated at specific loci within the catalytic core, their effect in promoting substrate promiscuity can be analysed in combination with the 3D model. K281I exerts a perturbation in the final access tunnel to the catalytic centre, generating a high (34‐fold) or mild (1.4‐fold) improvement in catalytic efficiency for FAE‐ and CE‐like substrates respectively (Fig. 4A and B). Saturation mutagenesis at the position 281 confirmed Ile as the best possible amino acid substitution for improving FAE phenotype. The Ile is a smaller residue making more accessible substrate channel that may correlate with higher values of kcat, whereas the substitution of a hydrophilic (Lys) by a hydrophobic (Ile) residue may correlate with enhanced binding of the substrates (lower Km values). This observation can also be extrapolated to the variant D282L (hydrophilic→hydrophobic); however, here, the slightly larger size of Asp than that of Leu appears to have more impact towards smaller substrates such as pNPC2 (Fig. 4C and D). Lys317His appear to have a significant negative impact on the properties of the enzyme since the mutation was associated with a complete loss of FAE activity. The effect on pNP esters was mostly associated with a twofold decrease in substrate affinity while slightly affecting the reaction rates (kcat). Since it was suggested that the presence of at least one m/p‐methoxy group in the cinnamate‐like substrates is required for binding (Tarbouriech et al., 2005), one could argue that the mutation at His317 may cause a partial unwinding of the loop and shortening the distance between binding residues and cinnamate‐like substrates while maintaining the activity with common substrates (Fig. 4E and F). The mutation Asn316STOP produced a deletion of the C‐terminal tail (25 amino acids) (Fig. 4G and H). This fragment constitutes an α‐helix located back to the catalytic cavity. Although, the deletion does not seem to be directly relevant to the catalytic core, we suggest that it may confer a higher flexibility, making catalysis more efficient, in particular, with shorter substrates. To further assess whether the higher flexibility is produced at expenses of lower structural stability, as suggested by the model, we studied the biophysical parameters of this protein variant after pre‐incubation with different concentrations of guanidinium chloride (GdmCl) and at different temperatures. We showed that this variant tends to misfold to a large degree after pre‐incubation with ≥ 0.64 M GdmCl whereas the wild‐type protein was mostly chemically stable (misfolding occurred ≥ 2.1 M GdmCl) (Fig. S7).

Bottom Line: Although, single to triple mutants with both improved activities (up to 180-fold in k(cat)/K(m) values) and enzymes with inverted specificity ((k(cat)/K(m))(CE)/(k(cat)/K(m))(FAE) ratio of ∼0.4) were identified, no CE inactive variant was found.Screening of a large error-prone PCR-generated library yielded by far less mutants for substrate discrimination.We also found that no significant changes in CE activation energy occurs after any mutation (7.3 to -5.6 J mol(-1)), whereas a direct correlation between loss/gain of FAE function and activation energies (from 33.05 to -13.7 J mol(-1)) was found.

View Article: PubMed Central - PubMed

Affiliation: CSIC, Institute of Catalysis, 28049 Madrid, Spain. CSIC, Instituto de Agroquímica y Tecnología de Alimentos, 46980 Valencia, Spain.

Show MeSH
Related in: MedlinePlus