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(Reference retrieved automatically from Web of Science through information on FAPESP grant and its corresponding number as mentioned in the publication by the authors.)

Crystal structure of beta 1 -> 6-galactosidase from Bifidobacterium bifidum S17: trimeric architecture, molecular determinants of the enzymatic activity and its inhibition by alpha-galactose

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Author(s):
Godoy, Andre Schutzer ; Camilo, Cesar Moises ; Kadowaki, Marco Antonio ; Muniz, Heloisa dos S. ; Santo, Melissa Espirito ; Murakami, Mario Tyago ; Nascimento, Alessandro S. ; Polikarpov, Igor
Total Authors: 8
Document type: Journal article
Source: FEBS Journal; v. 283, n. 22, p. 4097-4112, NOV 2016.
Web of Science Citations: 5
Abstract

In a search for better comprehension of beta-galactosidase function and specificity, we solved the crystal structures of the GH42 beta-galactosidase BbgII from Bifidobacterium bifidum S17, a well-adapted probiotic microorganism from the human digestive tract, and its complex with D-alpha-galactose. BbgII is a three-domain molecule that forms barrel-shaped trimers in solution. BbgII interactions with D-alpha-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 beta 1 -> 6 and beta 1 -> 3 bonds, while binding of the substrates with beta 1 -> 4 linkages is less favorable. Database Structural data are available in the PDB under the accession numbers 4UZS and 4UCF (AU)

FAPESP's process: 11/20505-4 - Two important classes of glycosyl hydrolases: functional studies and structural analysis
Grantee:Marco Antonio Seiki Kadowaki
Support type: Scholarships in Brazil - Post-Doctorate
FAPESP's process: 09/05328-9 - Expression, purification and cristalization studies of the ligand-binding domain of nuclear receptors PPAR-alpha e PPAR-gamma
Grantee:Andre Schutzer de Godoy
Support type: Scholarships in Brazil - Scientific Initiation
FAPESP's process: 08/56255-9 - Structure and function of enzymes and auxiliary proteins from Trichoderma, active in cell-wall hydrolysis
Grantee:Igor Polikarpov
Support type: Program for Research on Bioenergy (BIOEN) - Thematic Grants
FAPESP's process: 14/06565-2 - Extending the frontiers in biomolecular interactions: docking and free energy assessment
Grantee:Alessandro Silva Nascimento
Support type: Regular Research Grants
FAPESP's process: 09/52840-7 - Center of Biological and Industrial Processes for Biofuels - CeProBIO
Grantee:Igor Polikarpov
Support type: Program for Research on Bioenergy (BIOEN) - Thematic Grants
FAPESP's process: 11/05712-3 - Structural and functional studies of beta-galactosidases from Xanthomonas campestris
Grantee:Andre Schutzer de Godoy
Support type: Scholarships in Brazil - Doctorate (Direct)