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ProGlyProt ID
BC105
Organism Information
Organism Name
Bacillus lentus
Domain
Bacteria
Classification
Family: Bacillaceae
Order: Bacillales
Class: Bacilli (or Firmibacteria)
Division or phylum: "Firmicutes"
Taxonomic ID (NCBI)
 
 
Protein Information
Protein Name
Subtilisin (SBL)- Cys mutant
UniProtKB/SwissProt ID
UniProtKB Sequence
Sequence length
269 AA
Subcellular Location
Secreted
Function
Subtilisin is an extracellular alkaline serine protease. EC = 3.4.21.62
 
 
Protein Structure
Homology Model
Homology Model File
PDB ID
 
 
Glycosylation Status
Glycosylation Type
S (Cys) linked
Experimentally Validated Glycosite(s) in Full Length Protein
N62C, S156C, S166C and L217C (Residues that are shown glycosylated in vitro upon mutation from, N, S and L to Cys respectively)
Experimentally Validated Glycosite(s ) in Mature Protein
N62C, S156C, S166C and L217C (Residues that are shown glycosylated in vitro upon mutation from, N, S and L to Cys respectively)
Glycosite(s) Annotated Protein Sequence
>1JEA:A|PDBID|CHAIN|SEQUENCE AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFV
PGEPSTQDGNGHGTHVAGTIAALNNSIGVL GVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSL
GSPSPSATLEQAVNSATSRGVLVVAASGNSGAGSIS YPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVN
VQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQI RNHLKNTATSLGSTNLYGSGLVNAEAATR/> This sequence was mutated at four residues to cysteines which were then g
lycosylated: >1JEA:A|PDBID|CHAIN|SEQUENCE AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLD
TGISTHPDLNIRGGASFVPGEPSTQDGC*(62)GHGTHVAGTIAALNNSIGVL GVAPSAELYAVKVLGASGS
GSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNC*(156)GAGSIC*(166)
YPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASC*(217)NGTSMATPHVAGAA
ALVKQKNPSWSNVQI RNHLKNTATSLGSTNLYGSGLVNAEAATR
Sequence Around Glycosites (21 AA)
VPGEPSTQDGCGHGTHVAGTI
GVLVVAASGNCGAGSISYPAR
ASGNSGAGSICYPARYANAMA
STYPGSTYASCNGTSMATPHV
Technique(s) used for Glycosylation Detection
In vitro chemical glycosylation
Technique(s) used for Glycosylated Residue(s) Detection
Not applicable
Protein Glycosylation- Implication
In vitro engineered glycosylations of native subtilisin has been shown to affect structure as well as enzymatic activity.
 
 
Glycan Information
Glycan Annotation
L217C-S-β-Glc(Ac)2, L217C-S-β-Glc(Ac)3, N62C-S-β-Glc(Ac)4, S156C-S-β-Glc(Ac)4, S16C-S-β-Glc(Ac)4.
Technique(s) used for Glycan Identification
Acetylated glycans were predetermined and synthesized in vitro
 
 
Literature
Reference(s)
1) Davis, B.G., Lloyd, R.C. and Jones, J.B. (2000) Controlled site-selective protein glycosylation for precise glycan structure-catalytic activity relationships. Bioorg Med Chem, 8, 1527-1535. [PubMed: 10976501]
2) Lloyd, R.C. Davis, B.G. and Jones, J.B. (2000) Site-selective glycosylation of subtilisin Bacillus lentus causes dramatic increases in esterase activity. Bioorg Med Chem, 8, 1537-44. [PubMed: 10976502]
3) Graycar, T., Knapp, M., Ganshaw, G., Dauberman, J. and Bott, R. (1999) Engineered Bacillus lentus subtilisins having altered flexibility. J Mol Biol, 292, 97-109. [PubMed: 10493860]
Additional Comments
Engineered glycoprotein.
Site-selective cysteine (modified mutant) glycosylations of subtilisin, a Bacillus lentus (SBL) protein has been shown to affect structure as well as enzymatic activity post in vitro glycosylation. A positive correlation has also been derived between acetylation of glycan attached and the enzymatic activity and specificity against an esterase substrate succinyl-Ala-Ala-Pro-Phe-S-benzyl by glycosylated subtilisin.
It is one of the first examples of preparations of homogeneous neoglycoproteins in which both the glycosite and structure of the introduced glycan were predetermined. Glycans containing different numbers of acetate groups were introduced at selected cysteine residues of the the mutated protein using peracetylated MTS (methanethiosulfonate) reagents following a careful pH adjustment.
Year of Identification
2000
Year of Validation
2000
 
 
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