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Biochemical and structural characterization of quinoprotein aldose sugar dehydrogenase from Thermus thermophilus HJ6: Mutational analysis of Tyr156 in the substrate-binding site

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Title
Biochemical and structural characterization of quinoprotein aldose sugar dehydrogenase from Thermus thermophilus HJ6: Mutational analysis of Tyr156 in the substrate-binding site
Other Titles
Thermus thermophilus HJ6 유래의 알도우즈 당 탈수소 효소의 생화학적 특성 및 구조분석
Authors
Kim, Han-Woo
Jeon, Sung-Jong
Park, Hyun
Park, Ae Kyung
Lee, Ji-Yeon
Wang, Ji-Yeon
Keywords
Alcohol oxidation; Aldose sugar dehydrogenase; Soluble quinoprotein; glucose dehydrogenase
Issue Date
2016
Citation
Kim, Han-Woo, et al. 2016. "Biochemical and structural characterization of quinoprotein aldose sugar dehydrogenase from Thermus thermophilus HJ6: Mutational analysis of Tyr156 in the substrate-binding site". ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 608(1): 20-26.
Abstract
The gene encoding a quinoprotein aldose sugar dehydrogenase (ASD) from Thermus thermophilus HJ6 (Tt_ASD) was cloned and sequenced;it comprised 1059 nucleotides encoding a protein containing 352 amino acids that had a predicted molecular mass of 38.9 kDa. The deduced amino acid sequence showed 42.9% and 33.9% identities to the ASD proteins from Pyrobaculum aerophilum and Escherichia coli, respectively. The biochemical properties of Tt_ASD were characterized. The optimum pH for the oxidation of glucose was 7.0?7.5 and the optimum temperature was 70 °C. The half-life of heat inactivation for the apoenzyme was about 25 min at 85 °C. The enzyme was highly thermostable, and the activity of the pyrroloquinoline quinone-bound holoenzyme was not lost after incubation at 85 °C for 100 min. Tt_ASD could oxidize various sugars, including hexoses, pentoses, disaccharides, and polysaccharides, in addition to alcohols. Structural analysis suggested that Tyr156 would be the substrate-binding residue. Two mutants, Y156A and Y156K, had impaired activities and affinities for all substrates and completely lost their activities for alcohols. This structural and mutational analysis of Tt_ASD demonstrates the crucial role of Tyr156 in determining substrate specificity.
URI
http://repository.kopri.re.kr/handle/201206/7481
DOI
http://dx.doi.org/10.1016/j.abb.2016.08.022
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