title: Maltase protein of Ogataea (Hansenula) polymorpha is a counterpart to the resurrected ancestor protein ancMALS of yeast maltases and isomaltases.
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.
authors.affiliation: Department of Genetics, Institute of Molecular and Cell Biology, University of Tartu, Estonia.
authors.forename: Katrin
authors.initials: K
authors.lastname: Viigand
authors.affiliation: Department of Genetics, Institute of Molecular and Cell Biology, University of Tartu, Estonia.
authors.forename: Triinu
authors.initials: T
authors.lastname: Visnapuu
authors.affiliation: Department of Genetics, Institute of Molecular and Cell Biology, University of Tartu, Estonia.
authors.forename: Karin
authors.initials: K
authors.lastname: Mardo
authors.affiliation: Department of Genetics, Institute of Molecular and Cell Biology, University of Tartu, Estonia.
authors.forename: Anneli
authors.initials: A
authors.lastname: Aasamets
authors.affiliation: Department of Genetics, Institute of Molecular and Cell Biology, University of Tartu, Estonia.
authors.forename: Tiina
authors.initials: T
authors.lastname: Alamäe
mesh_terms: D019943:Amino Acid Substitution; D055162:Biocatalysis; D020134:Catalytic Domain; D002855:Chromatography, Thin Layer; D005656:Fungal Proteins / Q000737:chemistry* / Q000145:classification / Q000235:genetics; D017353:Gene Deletion; D005800:Genes, Fungal; D005947:Glucose / Q000378:metabolism; D006868:Hydrolysis; D009834:Oligo-1,6-Glucosidase / Q000737:chemistry* / Q000145:classification / Q000235:genetics; D010802:Phylogeny; D010843:Pichia / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity; D013912:Threonine / Q000235:genetics; D014633:Valine / Q000235:genetics; D000520:alpha-Glucosidases / Q000737:chemistry* / Q000145:classification / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D013912:Threonine; D009834:Oligo-1,6-Glucosidase; C424662:MAL1 protein, Pichia angusta; D000520:alpha-Glucosidases; D014633:Valine; D005947:Glucose
keywords: HpMAL1; differential scanning fluorimetry; maltase-isomaltase; methylotrophic yeast; protein evolution; α-glucosidase