title: A novel method of production and biophysical characterization of the catalytic domain of yeast oligosaccharyl transferase.
abstract: Oligosaccharyl transferase (OT) is a multisubunit enzyme that catalyzes N-linked glycosylation of nascent polypeptides in the lumen of the endoplasmic reticulum. In the case of Saccharomyces cerevisiae, OT is composed of nine integral membrane protein subunits. Defects in N-linked glycosylation cause a series of disorders known as congenital disorders of glycosylation (CDG). The C-terminal domain of the Stt3p subunit has been reported to contain the acceptor protein recognition site and/or catalytic site. We report here the subcloning, overexpression, and a robust but novel method of production of the pure C-terminal domain of Stt3p at 60-70 mg/L in Escherichia coli. CD spectra indicate that the C-terminal Stt3p is highly helical and has a stable tertiary structure in SDS micelles. The well-dispersed two-dimensional (1)H-(15)N HSQC spectrum in SDS micelles indicates that it is feasible to determine the atomic structure by NMR. The effect of the conserved D518E mutation on the conformation of the C-terminal Stt3p is particularly interesting. The replacement of a key residue, Asp(518), located within the WWDYG signature motif (residues 516-520), led to a distinct tertiary structure, even though both proteins have similar overall secondary structures, as demonstrated by CD, fluorescence and NMR spectroscopies. This observation strongly suggests that Asp(518) plays a critical structural role, in addition to the previously proposed catalytic role. Moreover, the activity of the protein was confirmed by saturation transfer difference and nuclear magnetic resonance titration studies.
authors.affiliation: Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama 36849, USA.
authors.forename: Chengdong
authors.initials: C
authors.lastname: Huang
authors.forename: Smita
authors.initials: S
authors.lastname: Mohanty
authors.forename: Monimoy
authors.initials: M
authors.lastname: Banerjee
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D006602:Hexosyltransferases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D008823:Micelles; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D008565:Membrane Proteins; D008823:Micelles; D021122:Protein Subunits; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D001224:Aspartic Acid; D012967:Sodium Dodecyl Sulfate; D018698:Glutamic Acid; D006602:Hexosyltransferases; C096554:STT3 protein, S cerevisiae