PDBsum entry: 1np0

Hydrolase

PDB id: 1np0

Contents

Protein chains

480 a.a. *

Ligands

NAG-NAG ×2

NGT ×2

GOL ×3

SO4

Waters ×244

* Residue conservation analysis

PDB id:

1np0

Name:

Hydrolase

Title:

Human lysosomal beta-hexosaminidase isoform b in complex wit intermediate analogue NAG-thiazoline

Structure:

Beta-hexosaminidase beta chain. Chain: a, b. Synonym: n-acetyl-beta-glucosaminidase, beta-n-acetylhexosa hexosaminidase b. Ec: 3.2.1.52

Source:

Homo sapiens. Human. Organism_taxid: 9606. Organ: placenta

Biol. unit:

Tetramer (from PDB file)

Resolution:

2.50Å R-factor: 0.200 R-free: 0.227

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

Date:

16-Jan-03 Release date: 29-Apr-03

Protein chains

P07686  (HEXB_HUMAN) -  Beta-hexosaminidase subunit beta

Seq: 556 a.a.

Struc: 480 a.a.

Enzyme reactions

• Enzyme class: E.C.3.2.1.52 - Beta-N-acetylhexosaminidase.
• Reaction: Hydrolysis of terminal non-reducing N-acetyl-D-hexosamine residues in N-acetyl-beta-D-hexosaminides.

 Gene Ontology (GO) functional annotation 

• Cellular component: membrane (4 terms)
• Biological process: metabolic process (25 terms)
• Biochemical function: catalytic activity (11 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.

ABSTRACT

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.