PDBsum entry 1nou

Go to PDB code: 
protein ligands Protein-protein interface(s) links
Hydrolase PDB id
Jmol PyMol
Protein chains
480 a.a. *
GOL ×3
Waters ×226
* Residue conservation analysis
PDB id:
Name: Hydrolase
Title: Native human lysosomal beta-hexosaminidase isoform b
Structure: Beta-hexosaminidase beta chain. Chain: a, b. Synonym: n-acetyl-beta-glucosaminidase, beta-n-acetylhexosa hexosaminidase b. Ec:
Source: Homo sapiens. Human. Organism_taxid: 9606. Organ: placenta
Biol. unit: Dimer (from PDB file)
2.40Å     R-factor:   0.203     R-free:   0.231
Authors: B.L.Mark,D.J.Mahuran,M.M.Cherney,D.Zhao,S.Knapp,M.N.G.James
Key ref:
B.L.Mark et al. (2003). Crystal structure of human beta-hexosaminidase B: understanding the molecular basis of Sandhoff and Tay-Sachs disease. J Mol Biol, 327, 1093-1109. PubMed id: 12662933 DOI: 10.1016/S0022-2836(03)00216-X
16-Jan-03     Release date:   08-Apr-03    
Go to PROCHECK summary

Protein chains
Pfam   ArchSchema ?
P07686  (HEXB_HUMAN) -  Beta-hexosaminidase subunit beta
556 a.a.
480 a.a.
Key:    PfamA domain  Secondary structure  CATH domain

 Enzyme reactions 
   Enzyme class: E.C.  - Beta-N-acetylhexosaminidase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]
      Reaction: Hydrolysis of terminal non-reducing N-acetyl-D-hexosamine residues in N-acetyl-beta-D-hexosaminides.
 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     extracellular region   9 terms 
  Biological process     metabolic process   30 terms 
  Biochemical function     hydrolase activity     7 terms  


DOI no: 10.1016/S0022-2836(03)00216-X J Mol Biol 327:1093-1109 (2003)
PubMed id: 12662933  
Crystal structure of human beta-hexosaminidase B: understanding the molecular basis of Sandhoff and Tay-Sachs disease.
B.L.Mark, D.J.Mahuran, M.M.Cherney, D.Zhao, S.Knapp, M.N.James.
In humans, two major beta-hexosaminidase isoenzymes exist: Hex A and Hex B. Hex A is a heterodimer of subunits alpha and beta (60% identity), whereas Hex B is a homodimer of beta-subunits. Interest in human beta-hexosaminidase stems from its association with Tay-Sachs and Sandhoff disease; these are prototypical lysosomal storage disorders resulting from the abnormal accumulation of G(M2)-ganglioside (G(M2)). Hex A degrades G(M2) by removing a terminal N-acetyl-D-galactosamine (beta-GalNAc) residue, and this activity requires the G(M2)-activator, a protein which solubilizes the ganglioside for presentation to Hex A. We present here the crystal structure of human Hex B, alone (2.4A) and in complex with the mechanistic inhibitors GalNAc-isofagomine (2.2A) or NAG-thiazoline (2.5A). From these, and the known X-ray structure of the G(M2)-activator, we have modeled Hex A in complex with the activator and ganglioside. Together, our crystallographic and modeling data demonstrate how alpha and beta-subunits dimerize to form either Hex A or Hex B, how these isoenzymes hydrolyze diverse substrates, and how many documented point mutations cause Sandhoff disease (beta-subunit mutations) and Tay-Sachs disease (alpha-subunit mutations).
  Selected figure(s)  
Figure 2.
Figure 2. Ribbon diagram of human b-hexosaminidase B. The b-subunits of the Hex B homodimer are colored with domain I in green and domain II in blue (the eight parallel strands of the b-barrel of domain II is colored sky blue). What appear to be common structural features of family 20 glycosidases is the absence of regular a-helices at positions a5 and a7 of the (b/a)[8]-barrel structure of domain II and an additional C-terminal helix following helix a8. This additional helix packs between domains I and II, spatially orienting the two domains relative to each other. Helix a7 consists of only two turns and is part of an extended loop that forms a major portion of the dimer interface. The subunits are related at the dimer interface by a crystallographic 2-fold symmetry axis running perpendicular to the page. The N and C termini created as a result of post-translational processing are numbered by residue. The labels N and C denote the extreme N (residue 55) and C (residue 552) termini visible within the electron density. The disulfide bonds Cys91-Cys137, Cys309-Cys360 and Cys534-Cys551 are drawn in brown, magenta and yellow, respectively. The analogue of the reaction intermediate NAG-thiazoline, bound in the active site of each subunit is drawn as a space-filling model with carbon atoms in gray, oxygen in red, nitrogen in blue and sulfur in yellow. The active sites of each subunit are located 37 Å apart. All ribbon diagrams were drawn with Molscript[82.] and rendered with Raster3D [83.] unless otherwise indicated.
Figure 6.
Figure 6. Predicted model of human Hex A-G[M2]-activator quaternary complex. (a and b) Two views of the predicted quaternary complex. Residues of the a-subunit identical to those of the b-subunit are colored blue, non-identical residues are colored light brown. Most of the conserved amino acids in the a and b-subunits are located in (b/a)[8]-barrel of domain II. The b-subunit is colored gray, with residues of the active site distinguished in orange. The G[M2]-activator protein complex (G[M2]-AP) docks into a large groove between the two subunits so that the terminal non-reducing GalNAc sugar on G[M2] can be presented to the a-subunit active site and removed. Two surface loops (magenta and green), present only on the a-subunit, interact with the docked activator protein and appear to be involved in creating a docking site unique to the a-subunit. The magenta colored loop is proteolytically removed from the b-subunit during post-translational processing and may represent a modification that regulates the metabolic function of this subunit. (c) Model of the GM2 oligosaccharide (yellow) bound to the a-subunit active site (gray). The distorted boat conformation of the terminal GalNAc to be removed (Gal, labeled in blue) and the pseudo-axial orientation of the scissile bond and leaving group are based on crystallographic observations of the Michaelis complex of chitobiose bound to SmCHB.[20.] By incorporating these conformational restraints into the model, only one reasonable position could be found for the sialic acid residue (labeled SIA) within the active site pocket. Once positioned, the negatively charged carboxylate of the sialic acid, which can only be accommodated by the a-subunit, was found to come within hydrogen bonding distance of Arg424, a positively charged residue that is unique to the a-subunit (the b-subunit contains a Leu at this position). aGlu394 and aAsn423 (which are both Asp residues in the b-subunit) are believed to help hold Arg424 into position. Arg424, in turn, stabilizes the negatively charged caboxylate of the sialic acid of the substrate via electrostatic and hydrogen-bonding interactions. The general acid-base residue, Glu323 (Glu355 in the b-subunit), can be seen interacting with the glycosidic oxygen atom of the scissile bond.
  The above figures are reprinted by permission from Elsevier: J Mol Biol (2003, 327, 1093-1109) copyright 2003.  
  Figures were selected by the author.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21373681 H.Usuki, Y.Yamamoto, Y.Kumagai, T.Nitoda, H.Kanzaki, and T.Hatanaka (2011).
MS/MS fragmentation-guided search of TMG-chitooligomycins and their structure-activity relationship in specific β-N-acetylglucosaminidase inhibition.
  Org Biomol Chem, 9, 2943-2951.  
20926324 J.T.Clarke, D.J.Mahuran, S.Sathe, E.H.Kolodny, B.A.Rigat, J.A.Raiman, and M.B.Tropak (2011).
An open-label Phase I/II clinical trial of pyrimethamine for the treatment of patients affected with chronic GM2 gangliosidosis (Tay-Sachs or Sandhoff variants).
  Mol Genet Metab, 102, 6.  
21290547 Y.Yang, T.Liu, Y.Yang, Q.Wu, Q.Yang, and B.Yu (2011).
Synthesis, Evaluation, and Mechanism of N,N,N-Trimethyl-D-glucosamine-(1→4)-chitooligosaccharides as Selective Inhibitors of Glycosyl Hydrolase Family 20 β-N-Acetyl-D-hexosaminidases.
  Chembiochem, 12, 457-467.  
21095575 H.C.Dorfmueller, V.S.Borodkin, M.Schimpl, X.Zheng, R.Kime, K.D.Read, and D.M.van Aalten (2010).
Cell-penetrant, nanomolar O-GlcNAcase inhibitors selective against lysosomal hexosaminidases.
  Chem Biol, 17, 1250-1255.
PDB code: 2xpk
20466648 K.Slámová, R.Gazák, P.Bojarová, N.Kulik, R.Ettrich, H.Pelantová, P.Sedmera, and V.Kren (2010).
4-Deoxy-substrates for beta-N-acetylhexosaminidases: how to make use of their loose specificity.
  Glycobiology, 20, 1002-1009.  
19917668 M.B.Tropak, S.W.Bukovac, B.A.Rigat, S.Yonekawa, W.Wakarchuk, and D.J.Mahuran (2010).
A sensitive fluorescence-based assay for monitoring GM2 ganglioside hydrolysis in live patient cells and their lysates.
  Glycobiology, 20, 356-365.  
20396401 T.M.Gloster, and D.J.Vocadlo (2010).
Mechanism, Structure, and Inhibition of O-GlcNAc Processing Enzymes.
  Curr Signal Transduct Ther, 5, 74-91.  
20445236 Z.S.Derewenda (2010).
Application of protein engineering to enhance crystallizability and improve crystal properties.
  Acta Crystallogr D Biol Crystallogr, 66, 604-615.  
19275764 H.C.Dorfmueller, V.S.Borodkin, M.Schimpl, and D.M.van Aalten (2009).
GlcNAcstatins are nanomolar inhibitors of human O-GlcNAcase inducing cellular hyper-O-GlcNAcylation.
  Biochem J, 420, 221-227.
PDB code: 2wb5
18484570 J.Intra, F.Cenni, G.Pavesi, M.Pasini, and M.E.Perotti (2009).
Interspecific analysis of the glycosidases of the sperm plasma membrane in Drosophila.
  Mol Reprod Dev, 76, 85.  
19499593 M.D.Balcewich, K.A.Stubbs, Y.He, T.W.James, G.J.Davies, D.J.Vocadlo, and B.L.Mark (2009).
Insight into a strategy for attenuating AmpC-mediated beta-lactam resistance: structural basis for selective inhibition of the glycoside hydrolase NagZ.
  Protein Sci, 18, 1541-1551.
PDB codes: 2wca 3gs6 3gsm
19442276 M.Pasztoi, G.Nagy, P.Geher, T.Lakatos, K.Toth, K.Wellinger, P.Pocza, B.Gyorgy, M.C.Holub, A.Kittel, K.Paloczy, M.Mazan, P.Nyirkos, A.Falus, and E.I.Buzas (2009).
Gene expression and activity of cartilage-degrading glycosidases in human rheumatoid arthritis and osteoarthritis synovial fibroblasts.
  Arthritis Res Ther, 11, R68.  
18473163 M.Wendeler, and K.Sandhoff (2009).
Hexosaminidase assays.
  Glycoconj J, 26, 945-952.  
18758829 S.Zampieri, M.Filocamo, E.Buratti, M.Stroppiano, K.Vlahovicek, N.Rosso, E.Bignulin, S.Regis, F.Carnevale, B.Bembi, and A.Dardis (2009).
Molecular and functional analysis of the HEXB gene in Italian patients affected with Sandhoff disease: identification of six novel alleles.
  Neurogenetics, 10, 49-58.  
18342252 D.G.Hogenkamp, Y.Arakane, K.J.Kramer, S.Muthukrishnan, and R.W.Beeman (2008).
Characterization and expression of the beta-N-acetylhexosaminidase gene family of Tribolium castaneum.
  Insect Biochem Mol Biol, 38, 478-489.  
18647384 J.Intra, G.Pavesi, and D.S.Horner (2008).
Phylogenetic analyses suggest multiple changes of substrate specificity within the glycosyl hydrolase 20 family.
  BMC Evol Biol, 8, 214.  
18469123 J.Wada, T.Ando, M.Kiyohara, H.Ashida, M.Kitaoka, M.Yamaguchi, H.Kumagai, T.Katayama, and K.Yamamoto (2008).
Bifidobacterium bifidum lacto-N-biosidase, a critical enzyme for the degradation of human milk oligosaccharides with a type 1 structure.
  Appl Environ Microbiol, 74, 3996-4004.  
18641620 P.M.Fischer (2008).
Turning down tau phosphorylation.
  Nat Chem Biol, 4, 448-449.  
17237499 G.H.Maegawa, M.Tropak, J.Buttner, T.Stockley, F.Kok, J.T.Clarke, and D.J.Mahuran (2007).
Pyrimethamine as a potential pharmacological chaperone for late-onset forms of GM2 gangliosidosis.
  J Biol Chem, 282, 9150-9161.  
17557860 H.Akeboshi, Y.Chiba, Y.Kasahara, M.Takashiba, Y.Takaoka, M.Ohsawa, Y.Tajima, I.Kawashima, D.Tsuji, K.Itoh, H.Sakuraba, and Y.Jigami (2007).
Production of recombinant beta-hexosaminidase A, a potential enzyme for replacement therapy for Tay-Sachs and Sandhoff diseases, in the methylotrophic yeast Ogataea minuta.
  Appl Environ Microbiol, 73, 4805-4812.  
17439950 K.A.Stubbs, M.Balcewich, B.L.Mark, and D.J.Vocadlo (2007).
Small molecule inhibitors of a glycoside hydrolase attenuate inducible AmpC-mediated beta-lactam resistance.
  J Biol Chem, 282, 21382-21391.
PDB code: 2oxn
17894780 M.B.Tropak, and D.Mahuran (2007).
Lending a helping hand, screening chemical libraries for compounds that enhance beta-hexosaminidase A activity in GM2 gangliosidosis cells.
  FEBS J, 274, 4951-4961.  
17317569 M.B.Tropak, J.E.Blanchard, S.G.Withers, E.D.Brown, and D.Mahuran (2007).
High-throughput screening for human lysosomal beta-N-Acetyl hexosaminidase inhibitors acting as pharmacological chaperones.
  Chem Biol, 14, 153-164.  
17509134 R.Ettrich, V.Kopecký, K.Hofbauerová, V.Baumruk, P.Novák, P.Pompach, P.Man, O.Plíhal, M.Kutý, N.Kulik, J.Sklenár, H.Ryslavá, V.Kren, and K.Bezouska (2007).
Structure of the dimeric N-glycosylated form of fungal beta-N-acetylhexosaminidase revealed by computer modeling, vibrational spectroscopy, and biochemical studies.
  BMC Struct Biol, 7, 32.  
17949435 S.G.Manuel, C.Ragunath, H.B.Sait, E.A.Izano, J.B.Kaplan, and N.Ramasubbu (2007).
Role of active-site residues of dispersin B, a biofilm-releasing beta-hexosaminidase from a periodontal pathogen, in substrate hydrolysis.
  FEBS J, 274, 5987-5999.  
17617724 T.Okada, S.Ishiyama, H.Sezutsu, A.Usami, T.Tamura, K.Mita, K.Fujiyama, and T.Seki (2007).
Molecular cloning and expression of two novel beta-N-acetylglucosaminidases from silkworm Bombyx mori.
  Biosci Biotechnol Biochem, 71, 1626-1635.  
  17015493 G.H.Maegawa, T.Stockley, M.Tropak, B.Banwell, S.Blaser, F.Kok, R.Giugliani, D.Mahuran, and J.T.Clarke (2006).
The natural history of juvenile or subacute GM2 gangliosidosis: 21 new cases and literature review of 134 previously reported.
  Pediatrics, 118, e1550-e1562.  
16698036 M.J.Lemieux, B.L.Mark, M.M.Cherney, S.G.Withers, D.J.Mahuran, and M.N.James (2006).
Crystallographic structure of human beta-hexosaminidase A: interpretation of Tay-Sachs mutations and loss of GM2 ganglioside hydrolysis.
  J Mol Biol, 359, 913-929.
PDB codes: 2gjx 2gk1
16478472 M.Wendeler, N.Werth, T.Maier, G.Schwarzmann, T.Kolter, M.Schoeniger, D.Hoffmann, T.Lemm, W.Saenger, and K.Sandhoff (2006).
The enzyme-binding region of human GM2-activator protein.
  FEBS J, 273, 982-991.  
16684772 N.Tomiya, S.Narang, J.Park, B.Abdul-Rahman, O.Choi, S.Singh, J.Hiratake, K.Sakata, M.J.Betenbaugh, K.B.Palter, and Y.C.Lee (2006).
Purification, characterization, and cloning of a Spodoptera frugiperda Sf9 beta-N-acetylhexosaminidase that hydrolyzes terminal N-acetylglucosamine on the N-glycan core.
  J Biol Chem, 281, 19545-19560.  
16565725 R.J.Dennis, E.J.Taylor, M.S.Macauley, K.A.Stubbs, J.P.Turkenburg, S.J.Hart, G.N.Black, D.J.Vocadlo, and G.J.Davies (2006).
Structure and mechanism of a bacterial beta-glucosaminidase having O-GlcNAcase activity.
  Nat Struct Mol Biol, 13, 365-371.
PDB codes: 2chn 2cho
16880605 T.Itakura, A.Kuroki, Y.Ishibashi, D.Tsuji, E.Kawashita, Y.Higashine, H.Sakuraba, S.Yamanaka, and K.Itoh (2006).
Inefficiency in GM2 ganglioside elimination by human lysosomal beta-hexosaminidase beta-subunit gene transfer to fibroblastic cell line derived from Sandhoff disease model mice.
  Biol Pharm Bull, 29, 1564-1569.  
15795231 M.S.Macauley, G.E.Whitworth, A.W.Debowski, D.Chin, and D.J.Vocadlo (2005).
O-GlcNAcase uses substrate-assisted catalysis: kinetic analysis and development of highly selective mechanism-inspired inhibitors.
  J Biol Chem, 280, 25313-25322.  
16230343 T.Kolter, F.Winau, U.E.Schaible, M.Leippe, and K.Sandhoff (2005).
Lipid-binding proteins in membrane digestion, antigen presentation, and antimicrobial defense.
  J Biol Chem, 280, 41125-41128.  
16212488 T.Kolter, and K.Sandhoff (2005).
Principles of lysosomal membrane digestion: stimulation of sphingolipid degradation by sphingolipid activator proteins and anionic lysosomal lipids.
  Annu Rev Cell Dev Biol, 21, 81.  
15232573 A.H.Futerman, and G.van Meer (2004).
The cell biology of lysosomal storage disorders.
  Nat Rev Mol Cell Biol, 5, 554-565.  
15505380 I.Sinici, M.B.Tropak, D.J.Mahuran, and H.A.Ozkara (2004).
Assessing the severity of the small inframe deletion mutation in the alpha-subunit of beta-hexosaminidase A found in the Turkish population by reproducing it in the more stable beta-subunit.
  J Inherit Metab Dis, 27, 747-756.  
14724290 M.B.Tropak, S.P.Reid, M.Guiral, S.G.Withers, and D.Mahuran (2004).
Pharmacological enhancement of beta-hexosaminidase activity in fibroblasts from adult Tay-Sachs and Sandhoff Patients.
  J Biol Chem, 279, 13478-13487.  
14728689 M.Wendeler, J.Hoernschemeyer, D.Hoffmann, T.Kolter, G.Schwarzmann, and K.Sandhoff (2004).
Photoaffinity labelling of the human GM2-activator protein. Mechanistic insight into ganglioside GM2 degradation.
  Eur J Biochem, 271, 614-627.  
15485660 M.Zarghooni, S.Bukovac, M.Tropak, J.Callahan, and D.Mahuran (2004).
An alpha-subunit loop structure is required for GM2 activator protein binding by beta-hexosaminidase A.
  Biochem Biophys Res Commun, 324, 1048-1052.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB code is shown on the right.