Crystal structure of β1→6-galactosidase from Bifidobacterium bifidum S17: trimeric architecture, molecular determinants of the enzymatic activity and its inhibition by α-galactose.
In a search for better comprehension of β-galactosidase function and specificity, we solved the crystal structures of the GH42 β-galactosidase BbgII from Bifidobacterium bifidum S17, a well-adapted probiotic microorganism from the human digestive tract, and its complex with d-α-galactose. BbgII is a three-domain molecule that forms barrel-shaped trimers in solution. BbgII interactions with d-α-galactose, a competitive inhibitor, showed a number of residues that are involved in the coordination of ligands. A combination of site-directed mutagenesis of these amino acid residues with enzymatic activity measurements confirmed that Glu161 and Glu320 are fundamental for catalysis and their substitution by alanines led to catalytically inactive mutants. Mutation Asn160Ala resulted in a two orders of magnitude decrease of the enzyme kcatwithout significant modification in its Km, whereas mutations Tyr289Phe and His371Phe simultaneously decreased kcatand increased Kmvalues. Enzymatic activity of Glu368Ala mutant was too low to be detected. Our docking and molecular dynamics simulations showed that the enzyme recognizes and tightly binds substrates with β1→6 and β1→3 bonds, while binding of the substrates with β1→4 linkages is less favorable.



Structural data are available in the PDB under the accession numbers 4UZS and 4UCF.
Departamento de Física em São Carlos, Universidade de São Paulo, Brazil.
Andre Schutzer
AS
Godoy
Centro de Tecnologia Canavieira, Piracicaba, Brazil.
Cesar Moises
CM
Camilo
Departamento de Física em São Carlos, Universidade de São Paulo, Brazil.
Marco Antonio
MA
Kadowaki
Departamento de Física em São Carlos, Universidade de São Paulo, Brazil.
Heloisa Dos S
HD
Muniz
Departamento de Física em São Carlos, Universidade de São Paulo, Brazil.
Melissa
M
Espirito Santo
Biosciences National Laboratory, National Center for Energy and Materials Research, Campinas, Brazil.
Mario Tyago
MT
Murakami
Departamento de Física em São Carlos, Universidade de São Paulo, Brazil.
Alessandro S
AS
Nascimento
Departamento de Física em São Carlos, Universidade de São Paulo, Brazil.
Igor
I
Polikarpov
D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000069985:Bifidobacterium bifidum / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis / Q000187:drug effects; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005690:Galactose / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D005696:Galactosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008968:Molecular Conformation; D056004:Molecular Dynamics Simulation; D020125:Mutation, Missense; D000072417:Protein Domains; D055503:Protein Multimerization; D013379:Substrate Specificity
D000596:Amino Acids; D001426:Bacterial Proteins; D005696:Galactosidases; D005690:Galactose

Bifidobacterium bifidum
; GH42 family; X-ray structure; beta-galactosidase; probiotic