PDBsum entry 6f4c

Hydrolase

PDB id: 6f4c

Contents

Protein chain

363 a.a.

Waters ×63

PDB id:

6f4c

Name:

Hydrolase

Title:

Nicotiana benthamiana alpha-galactosidase

Structure:

Alpha-galactosidase. Chain: b. Engineered: yes

Source:

Nicotiana benthamiana. Organism_taxid: 4100. Expressed in: nicotiana benthamiana. Expression_system_taxid: 4100

Resolution:

2.80Å R-factor: 0.214 R-free: 0.285

Authors:

K.Kytidou,J.M.F.G.Aerts,N.S.Pannu

Key ref:

K.Kytidou et al. (2018). Nicotiana benthamiana α-galactosidase A1.1 can functionally complement human α-galactosidase A deficiency associated with Fabry disease. J Biol Chem, 293, 10042-10058. PubMed id: 29674318 DOI: 10.1074/jbc.RA118.001774

Date:

29-Nov-17 Release date: 25-Apr-18

Protein chain

A0A384E148  (A0A384E148_NICBE) -  Alpha-galactosidase from Nicotiana benthamiana

Seq: 363 a.a.

Struc: 363 a.a.

Enzyme reactions

• Enzyme class: E.C.3.2.1.22 - alpha-galactosidase.
• Reaction: Melibiose + H₂O = galactose + glucose
• Cofactor: Mg(2+); NAD(+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1074/jbc.RA118.001774

J Biol Chem 293:10042-10058 (2018)

PubMed id: 29674318

Nicotiana benthamiana α-galactosidase A1.1 can functionally complement human α-galactosidase A deficiency associated with Fabry disease.

K.Kytidou, J.Beekwilder, M.Artola, E.van Meel, R.H.P.Wilbers, G.F.Moolenaar, N.Goosen, M.J.Ferraz, R.Katzy, P.Voskamp, B.I.Florea, C.H.Hokke, H.S.Overkleeft, A.Schots, D.Bosch, N.Pannu, J.M.F.G.Aerts.

ABSTRACT

α-Galactosidases (EC 3.2.1.22) are retaining glycosidases that cleave terminal α-linked galactose residues from glycoconjugate substrates. α-Galactosidases take part in the turnover of cell wall-associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals. Deficiency of human α-galactosidase A (α-Gal A) causes Fabry disease (FD), a heritable, X-linked lysosomal storage disorder, characterized by accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Current management of FD involves enzyme-replacement therapy (ERT). An activity-based probe (ABP) covalently labeling the catalytic nucleophile of α-Gal A has been previously designed to study α-galactosidases for use in FD therapy. Here, we report that this ABP labels proteins in Nicotiana benthamiana leaf extracts, enabling the identification and biochemical characterization of an N. benthamiana α-galactosidase we name here A1.1 (gene accession ID GJZM-1660). The transiently overexpressed and purified enzyme was a monomer lacking N-glycans and was active toward 4-methylumbelliferyl-α-d-galactopyranoside substrate (K_m = 0.17 mm) over a broad pH range. A1.1 structural analysis by X-ray crystallography revealed marked similarities with human α-Gal A, even including A1.1's ability to hydrolyze Gb3 and lyso-Gb3, which are not endogenous in plants. Of note, A1.1 uptake into FD fibroblasts reduced the elevated lyso-Gb3 levels in these cells, consistent with A1.1 delivery to lysosomes as revealed by confocal microscopy. The ease of production and the features of A1.1, such as stability over a broad pH range, combined with its capacity to degrade glycosphingolipid substrates, warrant further examination of its value as a potential therapeutic agent for ERT-based FD management.