ProGP197 (Subtilisin (SBL)-Cys mutant)
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| ProGP ID | ProGP197 (Subtilisin (SBL)-Cys mutant) |
| Validation Status | Characterized |
| Organism Information | |
| Organism Name | Bacillus lentus |
| Domain | Bacteria |
| Classification | Phylum : Firmicutes Class : Bacilli Orders : Bacillales Family : Bacillaceae Genus : Bacillus Species : lentus |
| Taxonomic ID (NCBI) | 1467 |
| Protein Information | |
| Protein Name | Subtilisin (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 Location | Secreted |
| 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 Protein | N62C, S156C, S166C and L217C (Residues that are shown glycosylated in vitro upon mutation from, N, S and L to C 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 C 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 |
| 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 |
| Protein Glycosylation linked (PGL) gene(s) | |
| Additional Comment | 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. |
| Literature | |
| Year of Identification | 2000 |
| Year of Identification Month Wise | 2000.09.10 |
| Year of Validation | 2000 |
| Reference | Davis, B.G., Lloyd, R.C. and Jones, J.B., 2000. Controlled site-selective protein glycosylation for precise glycan structure–catalytic activity relationships. Bioorganic & medicinal chemistry, 8(7), pp.1527-1535. |
| Corresponding Author | Benjamin G Davis |
| Contact | Department of Chemistry, University of Durham, UK. ben. |
| 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. Bioorganic & medicinal chemistry, 8(7), pp.1537-1544. |
| Corresponding Author | J Bryan Jones Benjamin G Davis |
| Contact | Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, ON, Canada M5S 3H6 |
| Reference | Graycar, T., Knapp, M., Ganshaw, G., Dauberman, J. and Bott, R., 1999. Engineered Bacillus lentus subtilisins having altered flexibility. Journal of molecular biology, 292(1), pp.97-109. |
| Corresponding Author | Richard Bott |
| Contact | Genencor International, 925 Page Mill Road, Palo Alto, CA 94304, USA. |