Biochemical and structural characterization of quinoprotein aldose sugar dehydrogenase from Thermus thermophilus HJ6: Mutational analysis of Tyr156 in the substrate-binding site.
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.
Division of Life Sciences, Korea Polar Research Institute (KOPRI), Korea University of Science and Technology, 26 Songdomirae-ro, Incheon, 21990, Republic of Korea; Department of Polar Sciences, Korea University of Science and Technology, 26 Songdomirae-ro, Incheon, 21990, Republic of Korea.
Han-Woo
HW
Kim
Department of Biotechnology & Bioengineering, Dong-Eui University, 176 Eomgwangno, Busanjin-gu, Busan, 47340, Republic of Korea.
Ji-Yeon
JY
Wang
Department of Biotechnology & Bioengineering, Dong-Eui University, 176 Eomgwangno, Busanjin-gu, Busan, 47340, Republic of Korea.
Ji-Yeon
JY
Lee
Division of Life Sciences, Korea Polar Research Institute (KOPRI), Korea University of Science and Technology, 26 Songdomirae-ro, Incheon, 21990, Republic of Korea.
Ae-Kyung
AK
Park
Division of Life Sciences, Korea Polar Research Institute (KOPRI), Korea University of Science and Technology, 26 Songdomirae-ro, Incheon, 21990, Republic of Korea; Department of Polar Sciences, Korea University of Science and Technology, 26 Songdomirae-ro, Incheon, 21990, Republic of Korea.
Hyun
H
Park
Department of Biotechnology & Bioengineering, Dong-Eui University, 176 Eomgwangno, Busanjin-gu, Busan, 47340, Republic of Korea; Department of Smart-Biohealth, Dong-Eui University, 176 Eomgwangno, Busanjin-gu, Busan, 47340, Republic of Korea. Electronic address: jeon.sj@deu.ac.kr.
Sung-Jong
SJ
Jeon
D001223:Aspartate-Semialdehyde Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D004252:DNA Mutational Analysis; D029968:Escherichia coli Proteins / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008968:Molecular Conformation; D009154:Mutation; D016366:Open Reading Frames; D045542:PQQ Cofactor / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology / Q000235:genetics*; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D045542:PQQ Cofactor; C514874:Asd protein, E coli; D001223:Aspartate-Semialdehyde Dehydrogenase
Alcohol oxidation; Aldose sugar dehydrogenase; Soluble quinoprotein glucose dehydrogenase; Thermus thermophilus