ProGP101 (H(A16-M) (1,3-1,4)-beta-glucanase)

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ProGP ID ProGP101 (H(A16-M) (1,3-1,4)-beta-glucanase)
Validation Status Characterized
Organism Information
Organism NamePaenibacillus (Bacillus) macerans and Bacillus amyloliquefaciens (velezensis)
Domain Bacteria
Classification Phylum : Firmicutes
Class : Bacilli
Orders : Bacillales
Family : Paenibacillaceae
Genus : Paenibacillus
Species : macerans
Taxonomic ID (NCBI) 44252
Genome Information
GenBank X55959
EMBL X55959
Protein Information
Protein NameH(A16-M) (1,3-1,4)-beta-glucanase
UniProtKB/SwissProt ID P23904
EMBL-CDSCAA39426.1
UniProtKB Sequence >sp|P23904|GUB_PAEMA Beta-glucanase OS=Paenibacillus macerans PE=1 SV=2 MKKKSCFTLVTTFAFSLIFSVSALAGSVFWEPLSYFNRSTWEKADGYSNGGVFNCTWRAN NVNFTNDGKLKLGLTSSAYNKFDCAEYRSTNIYGYGLYEVSMKPAKNTGIVSSFFTYTGP AHGTQWDEIDIEFLGKDTTKVQFNYYTNGVGGHEKVISLGFDASKGFHTYAFDWQPGYIK WYVDGVLKHTATANIPSTPGKIMMNLWNGTGVDDWLGSYNGANPLYAEYDWVKYTSN
Sequence length 237 AA
Subcellular LocationSecreted
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, 2AYH
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 ProteinN31, N185
Glycosite(s) Annotated Protein Sequence MKKKSCFTLVTTFAFSLIFSVSALAQTGGSFFEPFNSYNSGTWEKADGYSNGGVFN*(56)CTWR ANNVNFTNDGKLKLGLTSSAYNKFDCAEYRSTNIYGYGLYEVSMKPAKNTGIVSSFFTYT GPAHGTQWDEIDIEFLGKDTTKVQFNYYTNGVGGHEKVISLGFDASKGFHTYAFDWQPGY IKWYVDGVLKHTATANIPSTPGKIMMNLWN*(210)GTGVDDWLGSYNGANPLYAEYDWVKYTSN
This sequence has 16 N-terminal amino acids (colored red) derived from AMY. The remaining residues are from MAC.
Sequence Around Glycosites (21 AA) ADGYSNGGVFNCTWRANNVNF
TPGKIMMNLWNGTGVDDWLGS
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Technique(s) used for Glycosylation DetectionMass 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 Linkage: GlcNAc-N-Asn
Protein Glycosylation linked (PGL) gene(s)
Additional CommentEngineered 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.
Literature
Year of Identification1994
Year of Identification Month Wise1994.01.01
Year of Validation 1994
ReferenceHahn, 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‐β‐D‐Glucan 4‐Glucanohydrolase H (A16‐M). European journal of biochemistry, 232(3), pp.849-858.
ContactResearch group crystallography, Max Delbrück Center for Molecular Medicine, Berlin, Germany.
ReferenceMeldgaard, M., 1994. Different effects of N-glycosylation on the thermostability of highly homologous bacterial (1, 3-1, 4)-β-glucanases secreted from yeast. Microbiology, 140(1), pp.159-166.
Corresponding Author M Meldgaard
ContactDepartment of Physiology, Carlsberg Laboratory, Copenhagen, Denmark.
ReferenceHahn, 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. Proceedings of the National Academy of Sciences, 91(22), pp.10417-10421.
ContactResearch group crystallography, Max Delbrück Center for Molecular Medicine, Berlin, Germany.
ReferenceKeitel, T., Meldgaard, M. and Heinemann, U., 1994. Cation binding to a Bacillus (1, 3–1, 4)‐β‐glucanase Geometry, affinity and effect on protein stability. European Journal of Biochemistry, 222(1), pp.203-214.
Corresponding Author Udo Heinemann
ContactInstitute of Crystallography, Freie Universität Berlin, Germany
ReferenceOlsen, O. and Thomsen, K.K., 1991. Improvement of bacterial β-glucanase thermostability by glycosylation. Microbiology, 137(3), pp.579-585.
Corresponding Author Karl Kristian Thomsen
ContactCarlsberg Laboratory, Department of Physiology, Gamle Carlsbergvej 10, DK-2500 Copenhagen Valby, Denmark