Open AccessInfluence of Substrate Conformation on the Deglycosylation of Ribonuclease B by Recombinant Yeast Peptide: N ‐glycanase
Author(s) -
WANG Shengjun,
WANG Peng George,
QI Qingsheng
Publication year - 2007
Publication title -
acta biochimica et biophysica sinica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.771
H-Index - 57
eISSN - 1745-7270
pISSN - 1672-9145
DOI - 10.1111/j.1745-7270.2007.00244.x
Subject(s) - rnase p , biochemistry , recombinant dna , ribonuclease , chemistry , dithiothreitol , enzyme , escherichia coli , peptide , yeast , glycoprotein , substrate (aquarium) , endoplasmic reticulum , biology , rna , ecology , gene
Peptide: N ‐glycanase has been thought to be responsible for proteasome‐dependent degradation of misfolded glycoproteins translocated from the endoplasmic reticulum (ER) to the cytosol. Therefore, the enzyme was supposed to be able to distinguish between native and non‐native glycoproteins. In the present study, a recombinant, yeast peptide: N ‐glycanase, Png1p, was expressed in Escherichia coli as inclusion bodies and was purified, refolded and characterized. The results showed that the recombinant enzyme has a broad pH range adaptation, from pH 4.0 to pH 10.0, and has an optimum temperature of 30°C. This enzyme is a zinc metalloenzyme. Its activity was abolished with the addition of EDTA and not restored by adding metal ions. Furthermore, the deglycosylation efficiency of recombinant Png1p from E. coli was investigated with respect to the substrate conformation in vitro. When ribonuclease B (RNase B) was denatured at 60‐65 °C or by 40‐60 mM dithiothreitol, indicated by its obvious structural change and sharpest activity change, its deglycosylation by Png1p was most prominent. The deglycosylation efficiency of RNase B by Png1p was found to be related to its structural conformation and enzymatic activity. Edited by Minghua XU