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) 44252 Genome Sequence (s) EMBL X55959 Gene Information Gene Name NCBI Gene ID Protein Information Protein Name H(A16-M) (1,3-1,4)-beta-glucanase UniProtKB/SwissProt ID P23904 NCBI RefSeq EMBL-CDS CAA39426.1 UniProtKB Sequence >sp|P23904|GUB_PAEMA Beta-glucanase OS=Paenibacillus macerans PE=1 SV=2 MKKKSCFTLVTTFAFSLIFSVSALAGSVFWEPLSYFNRSTWEKADGYSNGGVFNCTWRAN NVNFTNDGKLKLGLTSSAYNKFDCAEYRSTNIYGYGLYEVSMKPAKNTGIVSSFFTYTGP AHGTQWDEIDIEFLGKDTTKVQFNYYTNGVGGHEKVISLGFDASKGFHTYAFDWQPGYIK WYVDGVLKHTATANIPSTPGKIMMNLWNGTGVDDWLGSYNGANPLYAEYDWVKYTSN 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 1CPM, 1GLH Glycosylation Status Glycosylation Type N (Asn) linked Experimentally Validated Glycosite(s) in Full Length Protein (Signal peptide: 1-23) N54, N208 Experimentally Validated Glycosite(s ) in Mature Protein N31, N185 Glycosite(s) Annotated Protein Sequence MKKKSCFTLVTTFAFSLIFSVSALAQTGGSFFEPFNSYNSGTWEKADGYSNGGVFN*(56)CTWR ANNVNFTNDGKLKLGLTSSAYNKFDCAEYRSTNIYGYGLYEVSMKPAKNTGIVSSFFTYT GPAHGTQWDEIDIEFLGKDTTKVQFNYYTNGVGGHEKVISLGFDASKGFHTYAFDWQPGY IKWYVDGVLKHTATANIPSTPGKIMMNLWN*(210)GTGVDDWLGSYNGANPLYAEYDWVKYTSN This sequence has 16 N-terminal amino acids (colored dark-green) derived from AMY. The remaining residues are from MAC. Sequence Around Glycosites (21 AA) ADGYSNGGVFNCTWRANNVNF TPGKIMMNLWNGTGVDDWLGS Glycosite Sequence Logo seqlogo 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. Glycan Information Glycan Annotation Technique(s) used for Glycan Identification Protein Glycosylation linked (PGL) gene(s) OST Gene Name OST NCBI Gene ID OST GenBank Gene Sequence OST Protein Name OST UniProtKB/ SwissProt ID OST NCBI RefSeq OST EMBL-CDS OST UniProtKB Sequence OST EC Number (BRENDA) OST Genome Context Characterized Accessory Gene(s) PGL Additional Links CAZy Literatures 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. 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 E Year of Identification 1994 Year of Validation 1994