Structural characterization of a xylanase from psychrophilic yeast by mass spectrometry.
The complete structural characterization of the xylanase, a glycoprotein constituted of 338 amino acids, from psychrophilic antarctic yeast Criptococcus albidus TAE85 was achieved both at the protein and carbohydrate level by exploiting mass spectrometric procedures. The verification of the primary structure, the definition of the S-S pattern, the assignment of glycosylation sites and the investigation of glycosylation pattern were performed. This analysis revealed the occurrence of N-glycosylation only at Asn254, modified by high-mannose structure; moreover the protein resulted to be O-glycosylated with GalGalNAc structures. The data obtained on both the N- and O-linked glycans in the cold xylanase constitute the first description of the glycosylation pattern in psychrophylic microorganisms and suggest that the glycosylation system in cold-adapted organisms might have similarities as well as differences with respect to mesophylic and thermophylic cells. The cysteine pairings were eventually identified as Cys173-Cys205 and Cys272-Cys278, with Cys89 showing a free thiol group. These data suggest that a common folding motif might occur within the entire xylanase family in which the second Cys is linked to the third one with the fourth and fifth joined together.
Amoresano
A
A
International Mass Spectrometry Facilities Centre, via Pansini 5, 80131 Naples, Italy.
Andolfo
A
A
Corsaro
M M
MM
Zocchi
I
I
Petrescu
I
I
Gerday
C
C
Marino
G
G
D000595:Amino Acid Sequence; D002236:Carbohydrate Conformation; D003454:Cryptococcus / Q000201:enzymology*; D004220:Disulfides / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry; D006031:Glycosylation; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000737:chemistry*
D004220:Disulfides; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase