RT Journal Article
T1 Influence of key residues on the heterologous extracellular production offungal ribonuclease U2 in the yeast Pichia pastoris
A1 Álvarez García, Elisa
A1 García Ortega, Lucía
A1 De los Ríos, Vivian
A1 Gavilanes, José G.
A1 Martínez Del Pozo, Álvaro
AB Ribonuclease U2, secreted by the smut fungus Ustilago sphaerogena, isa cyclizing ribonuclease that displays a rather unusual specificity within thegroup of microbial extracellular RNases, best represented by RNase T1.Superposition of the three-dimensional structures of RNases T1 and U2suggests that the RNase U2 His 101 would be the residue equivalent to theRNase T1 catalytically essential His 92. RNase U2 contains three disulfidebridges but only two of them are conserved among the family of fungalextracellular RNases. The non-conserved disulfide bond is established betweenCys residues 1 and 54. Mispairing of the disulfide network due to the presenceof two consecutive Cys residues (54 and 55) has been invoked to explain thepresence of wrongly folded RNase U2 species when produced in P. pastoris. Inorder to study both hypotheses, the RNase U2 H101Q and C1/54S variantshave been produced, purified, and characterized. The results obtained supportthe major conclusion that His 101 is required for proper protein folding whensecreted by the yeast P. pastoris. On the other hand, substitution of the first Cysresidue for Ser results in a mutant version which is more efficiently processed interms of a more complete removal of the yeast α-factor signal peptide. Inaddition, it has been shown that elimination of the Cys 1-Cys 54 disulfide bridgedoes not interfere with RNase U2 proper folding, generating a natively foldedbut much less stable protein.
SN 1096-0279
YR 2009
FD 2009
LK https://hdl.handle.net/20.500.14352/50358
UL https://hdl.handle.net/20.500.14352/50358
LA eng
NO Ministerio de Educación y Ciencia de España
DS Docta Complutense
RD 10 abr 2025