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

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

• 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: 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: 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: 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)
• 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: 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: 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