Potentsiaalsete replikatsioonis osalevate valkude deletsiooni mõju mitokondriaalse DNA stabiilsusele ja hulgale

Valgud Rpo41 (RNA polymerase), Mgm101 (mitochondrial genome maintenance) ja Mhr1 (mitochondrial homologous recombination) on vajalikud mitokondriaalse DNA (mtDNA) stabiilsuseks, kuna nende valkude deletsioon viib mtDNA kadumiseni või fragmenteerumiseni metsiktüüpi (rho+) rakkudes. Pagaripärmis toimuva mtDNA replikatsiooni kohta on välja pakutud kaks hüpoteesi: transkriptsioon-sõltuv ning rekombinatsioon-sõltuv replikatsioon. Mitokondriaalsel replikatsioonil osalevate valkude deletsioonist tingitud fenotüüpi saab uurida rho- pärmitüvedes, sest kirjanduse põhjal on teada, et rho- mutandid on võimelised säilitama mitokondriaalset genoomi erinevate mtDNA stabiilsuse eest vastutavate valkude puudumise korral. Käesolevas töös kasutati nende valkude deletsiooni mõju uurimiseks tüve HSρ- a1184

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

Genome of an early-diverged yeast Blastobotrys (Arxula) adeninivorans (Ba) encodes 88 glycoside hydrolases (GHs) including two α-glucosidases of GH13 family. One of those, the rna_ARAD1D20130g-encoded protein (BaAG2; 581 aa) was overexpressed in Escherichia coli, purified and characterized. We showed that maltose, other maltose-like substrates (maltulose, turanose, maltotriose, melezitose, malto-oligosaccharides of DP 4‒7) and sucrose were hydrolyzed by BaAG2, whereas isomaltose and isomaltose-like substrates (palatinose, α-methylglucoside) were not, confirming that BaAG2 is a maltase. BaAG2 was competitively inhibited by a diabetes drug acarbose (Ki = 0.8 µM) and Tris (Ki = 70.5 µM). BaAG2 was competitively inhibited also by isomaltose-like sugars and a hydrolysis product—glucose. At high maltose concentrations, BaAG2 exhibited transglycosylating ability producing potentially prebiotic di- and trisaccharides. Atypically for yeast maltases, a low but clearly recordable exo-hydrolytic activity on amylose, amylopectin and glycogen was detected. Saccharomyces cerevisiae maltase MAL62, studied for comparison, had only minimal ability to hydrolyze these polymers, and its transglycosylating activity was about three times lower compared to BaAG2. Sequence identity of BaAG2 with other maltases was only moderate being the highest (51%) with the maltase MalT of Aspergillus oryzae