RESUMO
Hydrolysis efficiency of ß-galactosidases is affected due to a strong inhibition by galactose, hampering the complete lactose hydrolysis. One alternative to reduce this inhibition is to perform mutations in the enzyme's active site. The aim of this study was to evaluate the effect of point mutations on the active site of different microbial ß-galactosidases, using computational techniques. The enzymes of Aspergillus niger (AnßGal), Aspergillus oryzae (AoßGal), Bacillus circulans (BcßGal), Bifidobacterium bifidum (BbßGal), and Kluyveromyces lactis (KlßGal) were used. The mutations were carried out in all residues that were up to 4.5 Å from the galactose/lactose molecules and binding energy was computed. The mutants Tyr96Ala (AnßGal), Asn140Ala and Asn199Ala (AoßGal), Arg111Ala and Glu355Ala (BcßGal), Arg122Ala and Phe358Ala (BbßGal), Tyr523Ala, Phe620Ala, and Trp582Ala (KlßGal) had the best results, with higher effect on galactose binding energy and lower effect on lactose affinity. To maximize enzyme reactions by reducing galactose affinity, double mutations were proposed for BcßGal, BbßGal, and KlßGal. The double mutations in BcßGal and BbßGal caused the highest reduction in galactose affinity, while no satisfactory results were observed to KlßGal. Using computational tools, mutants that reduced galactose affinity without significantly affecting lactose binding were proposed. The mutations proposed can be used to reduce the negative feedback process, improving the catalytic characteristics of ß-galactosidases and rendering them promising for industrial applications.
Assuntos
Galactose/química , Lactose/química , beta-Galactosidase/genética , Aspergillus niger/enzimologia , Aspergillus oryzae/enzimologia , Bacillus/enzimologia , Bifidobacterium bifidum/enzimologia , Catálise , Hidrólise , Cinética , Kluyveromyces/enzimologia , Mutação Puntual/genética , beta-Galactosidase/química , beta-Galactosidase/ultraestruturaRESUMO
In a search for better comprehension of ß-galactosidase function and specificity, we solved the crystal structures of the GH42 ß-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 kcat without significant modification in its Km , whereas mutations Tyr289Phe and His371Phe simultaneously decreased kcat and increased Km 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 ß1â6 and ß1â3 bonds, while binding of the substrates with ß1â4 linkages is less favorable. DATABASE: Structural data are available in the PDB under the accession numbers 4UZS and 4UCF.
Assuntos
Proteínas de Bactérias/metabolismo , Bifidobacterium bifidum/enzimologia , Galactose/metabolismo , Galactosidases/metabolismo , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bifidobacterium bifidum/genética , Sítios de Ligação/genética , Biocatálise/efeitos dos fármacos , Domínio Catalítico , Cristalografia por Raios X , Galactose/química , Galactose/farmacologia , Galactosidases/química , Galactosidases/genética , Cinética , Conformação Molecular , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Domínios Proteicos , Multimerização Proteica , Especificidade por SubstratoRESUMO
Given the current interest in second-generation biofuels, carbohydrate-active enzymes have become the most important tool to overcome the structural recalcitrance of the plant cell wall. While some glycoside hydrolase families have been exhaustively described, others remain poorly characterized, especially with regard to structural information. The family 43 glycoside hydrolases are a diverse group of inverting enzymes; the available structure information on these enzymes is mainly from xylosidases and arabinofuranosidase. Currently, only one structure of an exo-ß-1,3-galactanase is available. Here, the production, crystallization and structure determination of a putative exo-ß-1,3-galactanase from Bifidobacterium bifidum S17 (BbGal43A) are described. BbGal43A was successfully produced and showed activity towards synthetic galactosides. BbGal43A was subsequently crystallized and data were collected to 1.4â Å resolution. The structure shows a single-domain molecule, differing from known homologues, and crystal contact analysis predicts the formation of a dimer in solution. Further biochemical studies are necessary to elucidate the differences between BbGal43A and its characterized homologues.