Premium
Crystal structure of β1→6‐galactosidase from Bifidobacterium bifidum S17: trimeric architecture, molecular determinants of the enzymatic activity and its inhibition by α‐galactose
Author(s) -
Godoy Andre Schutzer,
Camilo Cesar Moises,
Kadowaki Marco Antonio,
Muniz Heloisa dos S.,
Espirito Santo Melissa,
Murakami Mario Tyago,
Nascimento Alessandro S.,
Polikarpov Igor
Publication year - 2016
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.13908
Subject(s) - bifidobacterium bifidum , enzyme kinetics , stereochemistry , chemistry , enzyme , docking (animal) , mutant , galactose , biochemistry , active site , homology modeling , non competitive inhibition , bifidobacterium , medicine , nursing , fermentation , lactobacillus , gene
In a search for better comprehension of β‐galactosidase function and specificity, we solved the crystal structures of the GH 42 β‐galactosidase Bbg II from Bifidobacterium bifidum S17 , a well‐adapted probiotic microorganism from the human digestive tract, and its complex with d ‐α‐galactose. Bbg II is a three‐domain molecule that forms barrel‐shaped trimers in solution. Bbg II 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 k cat without significant modification in its K m , whereas mutations Tyr289Phe and His371Phe simultaneously decreased k cat and increased K m values. 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. Database Structural data are available in the PDB under the accession numbers 4UZS and 4UCF