| Texto completo | |
| Autor(es): |
Godoy, Andre Schutzer
;
Camilo, Cesar Moises
;
Kadowaki, Marco Antonio
;
Muniz, Heloisa dos S.
;
Santo, Melissa Espirito
;
Murakami, Mario Tyago
;
Nascimento, Alessandro S.
;
Polikarpov, Igor
Número total de Autores: 8
|
| Tipo de documento: | Artigo Científico |
| Fonte: | FEBS Journal; v. 283, n. 22, p. 16-pg., 2016-11-01. |
| Resumo | |
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) | |
| Processo FAPESP: | 11/05712-3 - Estudos estruturais e funcionais das enzimas beta-galactosidases de Xanthomonas campestris |
| Beneficiário: | Andre Schutzer de Godoy |
| Modalidade de apoio: | Bolsas no Brasil - Doutorado Direto |
| Processo FAPESP: | 09/52840-7 - Centro de Processos Biológicos e Industriais para Biocombustíveis - CeProBIO |
| Beneficiário: | Igor Polikarpov |
| Modalidade de apoio: | Auxílio à Pesquisa - Programa BIOEN - Temático |
| Processo FAPESP: | 11/20505-4 - Duas classes importantes de glicosil hidrolases: estudos funcionais e análise estrutural |
| Beneficiário: | Marco Antonio Seiki Kadowaki |
| Modalidade de apoio: | Bolsas no Brasil - Pós-Doutorado |
| Processo FAPESP: | 14/06565-2 - Estendendo as fronteiras em interações biomoleculares: docking e avaliação da energia livre |
| Beneficiário: | Alessandro Silva Nascimento |
| Modalidade de apoio: | Auxílio à Pesquisa - Regular |
| Processo FAPESP: | 08/56255-9 - Structure and function of enzymes and auxiliary proteins from Trichoderma, active in cell-wall hydrolysis |
| Beneficiário: | Igor Polikarpov |
| Modalidade de apoio: | Auxílio à Pesquisa - Programa BIOEN - Temático |
| Processo FAPESP: | 09/05328-9 - Expressão, purificação e ensaios de cristalização dos domínios de ligação ao ligante dos receptores nucleares PPAR-alfa e PPAR-gamma |
| Beneficiário: | Andre Schutzer de Godoy |
| Modalidade de apoio: | Bolsas no Brasil - Iniciação Científica |