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ProGlyProt ID
BC160
Organism Information
Organism Name
Paenibacillus (Bacillus) macerans and Bacillus amyloliquefaciens (velezensis)
Domain
Bacteria
Classification
Family: Bacillaceae
Order: Bacillales
Class: Bacilli (or Firmibacteria)
Division or phylum: "Firmicutes"
Taxonomic ID (NCBI)
 
 
Genome Sequence(s)
EMBL
 
 
Protein Information
Protein Name
H(A16-M) (1,3-1,4)-beta-glucanase
UniProtKB/SwissProt ID
P23904
EMBL-CDS
UniProtKB Sequence
Sequence length
237 AA
Subcellular Location
Secreted
Function
It is the hybrid enzyme that catalyses cleavage of (1,4)-beta-linkages of O-substituted beta-D-glucanopyranosyl residues.
 
 
Protein Structure
PDB ID
 
 
Glycosylation Status
Glycosylation Type
N (Asn) linked
Experimentally Validated Glycosite(s) in Full Length Protein
(Signal peptide: 1-25) N56, N210
Experimentally Validated Glycosite(s ) in Mature Protein
N31, N185
Glycosite(s) Annotated Protein Sequence
MKKKSCFTLVTTFAFSLIFSVSALAQTGGSFFEPFNSY
NSG
TWEKADGYSNGGVFN*(56)CTWR ANNVNFTNDGKLKLGLTSSAYNKFDCAEYRSTNIYGYGLYEVSM
KPAKNTGIVSSFFTYT GPAHGTQWDEIDIEFLGKDTTKVQFNYYTNGVGGHEKVISLGFDASKGFHTYAFDWQP
GY IKWYVDGVLKHTATANIPSTPGKIMMNLWN*(210)GTGVDDWLGSYNGANPLYAEYDWVKYTSN

This sequence has 16 N-terminal amino acids (colored magenta) derived fr
om AMY. The remaining residues are from MAC.
Sequence Around Glycosites (21 AA)
ADGYSNGGVFNCTWRANNVNF
TPGKIMMNLWNGTGVDDWLGS
Glycosite Sequence Logo
Glycosite Sequence Logo
Technique(s) used for Glycosylation Detection
Mass shift detected after deglycosylation with Endo Hf and PNGase F
Technique(s) used for Glycosylated Residue(s) Detection
N-terminal sequencing
Protein Glycosylation- Implication
There is a large influence of N glycosylation on the thermostability of the hybrid enzyme. 16- and 133-fold decrease of thermostability has been observed after treatment with endoglycosidase H and peptide:N-glycosidase F (that remove glycans). This indicates that N-glycans are a major determinant for the resistance of this hybrid glucanase to thermal inactivation.
 
 
Literature
Reference(s)
1) Hahn, M., Keitel, T. and Heinemann, U. (1995) Crystal and molecular structure at 0.16-nm resolution of the hybrid Bacillus endo-1,3-1,4-beta-D-glucan 4-glucanohydrolase H(A16-M). Eur J Biochem, 232, 849-858. [PubMed: 7588726]
2) Hahn, M., Piotukh, K., Borriss, R. and Heinemann, U. (1994) Native-like in vivo folding of a circularly permuted jellyroll protein shown by crystal structure analysis. Proc Natl Acad Sci U S A, 91, 10417-10421. [PubMed: 7937966]
3) Keitel, T., Meldgaard, M. and Heinemann, U. (1994) Cation binding to a Bacillus (1,3-1,4)-beta-glucanase. Geometry, affinity and effect on protein stability. Eur J Biochem, 222, 203-214. [PubMed: 8200344]
4) Meldgaard, M. and Svendsen, I. (1994) Different effects of N-glycosylation on the thermostability of highly homologous bacterial (1,3-1,4)-beta-glucanases secreted from yeast. Microbiology, 140 ( Pt 1), 159-166. [PubMed: 8162185]
5) Olsen, O., Thomesen, K. K. (1991) Improvement of bacterial P-glucanase thermostability by glycosylation. J Gen Microbiol, 137, 579–585.
Additional Comments
Engineered glycoprotein
The glucanases from which the hybrid enzyme is made, are not glycosylated in their native hosts. Hybrid enzyme containing 16 N-terminal amino acids from Bacillus amyloliquefaciens (1,3-1,4)-beta-glucanase (AMY), and remaining C-terminal part and signal peptide from B. macerans (1,3-1,4)-beta-glucanase (MAC), was expressed in Saccharomyces cerevisiae to see the effect of N-glycosylation on the thermostability of the hybrid enzyme.
Information from UniProtKB and EMBL has been provided for MAC enzyme as it constitutes the major part of the hybrid enzyme (239 AA). UniProtKB ID for AMY is P07980.
Year of Identification
1994
Year of Validation
1994
 
 
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