ProGP198

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ProGP ID ProGP198
Validation Status Characterized
Organism Information
Organism NameBacillus lentus
Domain Bacteria
Classification Family: Bacillaceae
Order: Bacillales
Class: Bacilli (or Firmibacteria)
Division or phylum: "Firmicutes"
Taxonomic ID (NCBI) 1467
Protein Information
Protein NameSubtilisin (SBL)- Cys mutant
UniProtKB/SwissProt ID  P29600
UniProtKB Sequence >sp|P29600|SUBS_BACLE Subtilisin Savinase OS=Bacillus lentus PE=1 SV=1 AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGN GHGTHVAGTIAALNNSIGVLGVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVA NLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGAGSISYPARYANAMAVGATDQNNNR ASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQI RNHLKNTATSLGSTNLYGSGLVNAEAATR
Sequence length 269 AA
Subcellular LocationSecreted
Function Subtilisin is an extracellular alkaline serine protease. EC = 3.4.21.62
Protein Structure
PDB ID 1JEA
Glycosylation Status
Glycosylation Type S (Cys) linked
Experimentally Validated Glycosite(s) in Full Length ProteinN62C, 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 ProteinN62C, 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 AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGNGHGTHVAGTIAALNNSIGVL GVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNSGAGSIS YPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASLNGTSMATPHVAGAAALVKQKNPSWSNVQI RNHLKNTATSLGSTNLYGSGLVNAEAATR
This sequence was mutated at four residues to cysteines which were then glycosylated: >1JEA:A|PDBID|CHAIN|SEQUENCE AQSVPWGISRVQAPAAHNRGLTGSGVKVAVLDTGISTHPDLNIRGGASFVPGEPSTQDGC*(62)GHGTHVAGTIAALNNSIGVL GVAPSAELYAVKVLGASGSGSVSSIAQGLEWAGNNGMHVANLSLGSPSPSATLEQAVNSATSRGVLVVAASGNC*(156)GAGSIC*(166) YPARYANAMAVGATDQNNNRASFSQYGAGLDIVAPGVNVQSTYPGSTYASC*(217)NGTSMATPHVAGAAALVKQKNPSWSNVQI RNHLKNTATSLGSTNLYGSGLVNAEAATR
Sequence Around Glycosites (21 AA) VPGEPSTQDGCGHGTHVAGTI
GVLVVAASGNCGAGSISYPAR
ASGNSGAGSICYPARYANAMA
STYPGSTYASCNGTSMATPHV
Glycosite Sequence Logo
Technique(s) used for Glycosylation DetectionIn 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
Additional CommentEngineered 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 Identification2000
Year of Identification Month Wise2000.09.10
Year of Validation 2000
Reference1) 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]
AuthorDavis, B.G., Lloyd, R.C. and Jones, J.B.
Research GroupDepartment of Chemistry, University of Durham, UK. ben.davis@durham.ac.uk
Corresponding Author Jones, J.B
ContactDepartment of Chemistry, University of Durham, UK. ben.davis@durham.ac.uk
Reference 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]
Author Bott, R.
Research GroupGenencor International, 925 Page Mill Road, Palo Alto, CA 94304, USA.
Corresponding Author Graycar, T., Knapp, M., Ganshaw, G., Dauberman, J. Bott, R.
ContactGenencor International, 925 Page Mill Road, Palo Alto, CA 94304, USA.
Reference 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]
Author Lloyd, R.C. Davis, B.G. Jones, J.B.
Research GroupDepartment of Chemistry, University of Toronto, ON, Canada.
Corresponding Author Jones, J.B.
ContactDepartment of Chemistry, University of Toronto, ON, Canada.
Reference 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]
Author Lloyd, R.C. Davis, B.G. and Jones, J.B
Research GroupDepartment of Chemistry, University of Toronto, ON, Canada.
Corresponding Author Jones, J.B.
ContactDepartment of Chemistry, University of Toronto, ON, Canada.