Influence of key residues on the heterologous extracellular production of fungal ribonuclease U2 in the yeast Pichia pastoris.
Ribonuclease U2, secreted by the smut fungus Ustilago sphaerogena, is a cyclizing ribonuclease that displays a rather unusual specificity within the group of microbial extracellular RNases, best represented by RNase T1. Superposition of the three-dimensional structures of RNases T1 and U2 suggests that the RNase U2 His 101 would be the residue equivalent to the RNase T1 catalytically essential His 92. RNase U2 contains three disulfide bridges but only two of them are conserved among the family of fungal extracellular RNases. The non-conserved disulfide bond is established between Cys residues 1 and 54. Mispairing of the disulfide network due to the presence of two consecutive Cys residues (54 and 55) has been invoked to explain the presence of wrongly folded RNase U2 species when produced in Pichia pastoris. In order to study both hypotheses, the RNase U2 H101Q and C1/54S variants have been produced, purified, and characterized. The results obtained support the major conclusion that His 101 is required for proper protein folding when secreted by the yeast P. pastoris. On the other hand, substitution of the first Cys residue for Ser results in a mutant version which is more efficiently processed in terms of a more complete removal of the yeast alpha-factor signal peptide. In addition, it has been shown that elimination of the Cys 1-Cys 54 disulfide bridge does not interfere with RNase U2 proper folding, generating a natively folded but much less stable protein.
Alvarez-García
Elisa
E
Departamento de Bioquímica y Biología Molecular I, Facultad de Ciencias Químicas, Universidad Complutense, Avenida Complutense s/n, Ciudad Universitaria, 28040 Madrid, Spain.
García-Ortega
Lucía
L
De los Ríos
Vivian
V
Gavilanes
José G
JG
Martínez-del-Pozo
Alvaro
A
D015153:Blotting, Western; D003545:Cysteine / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000378:metabolism*; D008958:Models, Molecular; D010843:Pichia / Q000235:genetics*; D017510:Protein Folding; D012694:Serine / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D014588:Ustilago / Q000201:enzymology* / Q000235:genetics
D012694:Serine; D006639:Histidine; D004722:Endoribonucleases; C022544:ribonuclease U2; D003545:Cysteine