ABSTRACT
Saccharomyces cerevisiae maltases use maltose, maltulose, turanose and maltotriose as substrates, isomaltases use isomaltose, α-methylglucoside and palatinose and both use sucrose. These enzymes are hypothesized to have evolved from a promiscuous α-glucosidase ancMALS through duplication and mutation of the genes. We studied substrate specificity of the maltase protein MAL1 from an earlier diverged yeast, Ogataea polymorpha (Op), in the light of this hypothesis. MAL1 has extended substrate specificity and its properties are strikingly similar to those of resurrected ancMALS. Moreover, amino acids considered to determine selective substrate binding are highly conserved between Op MAL1 and ancMALS. Op MAL1 represents an α-glucosidase in which both maltase and isomaltase activities are well optimized in a single enzyme. Substitution of Thr200 (corresponds to Val216 in S. cerevisiae isomaltase IMA1) with Val in MAL1 drastically reduced the hydrolysis of maltose-like substrates (α-1,4-glucosides), confirming the requirement of Thr at the respective position for this function. Differential scanning fluorimetry (DSF) of the catalytically inactive mutant Asp199Ala of MAL1 in the presence of its substrates and selected monosaccharides suggested that the substrate-binding pocket of MAL1 has three subsites (-1, +1 and +2) and that binding is strongest at the -1 subsite. The DSF assay results were in good accordance with affinity (Km ) and inhibition (Ki ) data of the enzyme for tested substrates, indicating the power of the method to predict substrate binding. Deletion of either the maltase (MAL1) or α-glucoside permease (MAL2) gene in Op abolished the growth of yeast on MAL1 substrates, confirming the requirement of both proteins for usage of these sugars. © 2016 The Authors. Yeast published by John Wiley & Sons, Ltd.
Subject(s)
Fungal Proteins/chemistry , Oligo-1,6-Glucosidase/chemistry , Pichia/enzymology , alpha-Glucosidases/chemistry , Amino Acid Substitution , Biocatalysis , Catalytic Domain , Chromatography, Thin Layer , Fungal Proteins/classification , Fungal Proteins/genetics , Gene Deletion , Genes, Fungal , Glucose/metabolism , Hydrolysis , Oligo-1,6-Glucosidase/classification , Oligo-1,6-Glucosidase/genetics , Phylogeny , Pichia/genetics , Pichia/growth & development , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Sequence Alignment , Substrate Specificity , Threonine/genetics , Valine/genetics , alpha-Glucosidases/classification , alpha-Glucosidases/geneticsSubject(s)
Genes, Fungal , Oligo-1,6-Glucosidase/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , alpha-Glucosidases/genetics , Chromosome Mapping , Chromosomes, Fungal/genetics , Multigene Family , Oligo-1,6-Glucosidase/classification , Open Reading Frames , Phylogeny , Saccharomyces cerevisiae Proteins/classification , alpha-Glucosidases/classificationABSTRACT
The alpha-amylase enzyme family is the largest family of glycoside hydrolases. It contains almost 30 different enzyme specificities covering hydrolases, transferases and isomerases. Some of the enzyme specificities from the family are closely related, others less so. This study, based on the analysis of 79 amino acid sequences, postulates two subfamilies in the framework of the aamylase family: the oligo-1,6-glucosidase subfamily and the neopullulanase subfamily. The specific sequence in the fifth conserved sequence region of the family served as the basis for defining the subfamilies: QpDln for the oligo-1,6-glucosidase subfamily and MPKln for the neopullulanase subfamily. This conserved sequence region is proposed to be the selection marker that enables one to distinguish between the two subfamilies. The 'intermediary' sequence MPDLN can be characteristic of the so-called intermediary group with a mixed enzyme specificity of alpha-amylase, cyclomaltodextrinase and neopullulanase. The evolutionary trees clearly supported the proposed definition of the two subfamilies.
