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 (K m) and inhibition (K i) 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.

A Highly Active Endo-Levanase BT1760 of a Dominant Mammalian Gut Commensal Bacteroides thetaiotaomicron Cleaves Not Only Various Bacterial Levans, but Also Levan of Timothy Grass

“…When compared with T m (65.4°C) of the levansucrase Lsc3 of Pseudomonas syringae pv. tomato [44] that was applied in this study for levan synthesis, the respective T m of BT1760 was considerably lower being comparable with respective value of the maltase protein of a yeast Ogataea polymorpha [68]. Notably, Lsc3 of P .…”

Characterization of a Maltase from an Early-Diverged Non-Conventional Yeast Blastobotrys adeninivorans

“…In BaAG2, a Thr is present at position of Val216 and therefore we predicted that this enzyme is most probably a maltase. However, as maltase-isomaltases also have a Thr at that position ( Figure 1, upper panel; [12,16]), based on the amino acid signature, BaAG2 may also be a promiscuous enzyme with a wide substrate spectrum like O. polymorpha MAL1. Figure 2 presents fragments of sequence comparison of BaAG2 with those of five experimentally studied maltases from GH13 family: two from bacteria, two from yeasts and one from a filamentous fungus Aspergillus.…”

“…In the genomes of most yeasts addressed in Viigand et al [12], the α-glucosidase genes resided in maltose utilization (MAL) clusters, whereas no MAL clusters were detected in B. adeninivorans. α-Glucosidases have been popular objects to study protein evolution and phylogenesis [13][14][15][16], but they also have a biotechnological value. Thus, some of them have a high transglycosylating activity thanks to which they produce prebiotic oligosaccharides and potential functional food ingredients such as panose, melezitose, isomelezitose and isomalto-oligosaccharides [17][18][19][20][21].…”

“…Maltases degrade maltose and maltose-like sugars, i.e., maltotriose, turanose and maltulose, but cannot degrade isomaltose and isomaltose-like sugars (α-1,6 linkages) such as palatinose. Both types of enzymes hydrolyze sucrose and a synthetic substrate p-nitrophenyl-α-d-glucopyranoside (pNPG) [15,16,23,24]. At the same time, some yeasts such as Ogataea polymorpha and Scheffersomyces stipitis, have promiscuous maltase-isomaltases that hydrolyze maltose-and isomaltose-like substrates [12,16,25].…”

Investigating cross‐contamination by yeast strains from dental solid waste to waste‐handling workers by DNA sequencing

“…The authors also compared culturing and DNA sequencing techniques to identify fungal species and suggested that cloning, cultivation, and quantitative PCR methods complemented each other, generating a more comprehensive picture of fungal content. The literature attested that the methylotrophic yeast O. polymorpha is one of the most important industrially applied yeast and could be isolated among others from soil, spoiled orange juice, leaf surfaces, exudates of plants, and insect guts (Suh & Zhou, ; Viigand, Visnapuu, Mardo, Aasamets, & Alamäe, ). The recovery of yeast species from waste workers' hands, nasal mucosa, and professional coats reinforces the need of frequent training programs inside health care institutions.…”