Crystal structure of thermophilic dextranase from Thermoanaerobacter pseudethanolicus.
The crystal structures of the wild type and catalytic mutant Asp-312→Gly in complex with isomaltohexaose of endo-1,6-dextranase from the thermophilic bacterium Thermoanaerobacter pseudethanolicus (TpDex), belonging to the glycoside hydrolase family 66, were determined. TpDex consists of three structural domains, a catalytic domain comprising an (β/α)8-barrel and two β-domains located at both N- and C-terminal ends. The isomaltohexaose-complex structure demonstrated that the isomaltohexaose molecule was bound across the catalytic site, showing that TpDex had six subsites (-4 to +2) in the catalytic cleft. Marked movement of the Trp-376 side-chain along with loop 6, which was the side wall component of the cleft at subsite +1, was observed to occupy subsite +1, indicating that it might expel the cleaved aglycone subsite after the hydrolysis reaction. Structural comparison with other mesophilic enzymes indicated that several structural features of TpDex, loop deletion, salt bridge and surface-exposed charged residue, may contribute to thermostability.
Biomolecular Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba 305-8602, Japan; zui@affrc.go.jp u9897854@jnu.ac.kr.
Nobuhiro
N
Suzuki
Biomolecular Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba 305-8602, Japan; zui@affrc.go.jp u9897854@jnu.ac.kr.
Naomi
N
Kishine
Biomolecular Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba 305-8602, Japan; zui@affrc.go.jp.
Zui
Z
Fujimoto
Biomolecular Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba 305-8602, Japan;
Mutsumi
M
Sakurai
Biomolecular Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba 305-8602, Japan;
Mitsuru
M
Momma
Eco-Friendly Bio-material Research Center, Korea Research Institute of Bioscience and Biotechnology, Jeongeup 580-185, Korea;
Jin-A
JA
Ko
Department of Food Science and Technology and Functional Food Research Center, Chonnam National University, Gwangju 500-757, Korea;
Seung-Hee
SH
Nam
Division of Applied Bioscience, Research Faculty of Agriculture, Hokkaido University, Kita-9 Nisi-9, Kita-ku, Sapporo 060-8589, Japan;
Atsuo
A
Kimura
Department of Food Science and Technology and Functional Food Research Center, Chonnam National University, Gwangju 500-757, Korea; Bioenergy Research Center, Chonnam National University, Gwangju 500-757, Korea u9897854@jnu.ac.kr.
Young-Min
YM
Kim
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003910:Dextranase / Q000737:chemistry* / Q000235:genetics; D004795:Enzyme Stability; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000737:chemistry*; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D046968:Thermoanaerobacter / Q000201:enzymology*
D001426:Bacterial Proteins; D009844:Oligosaccharides; C024046:isomaltohexose; D003910:Dextranase
Thermoanaereobacter; dextranase; enzyme–substrate complex; glycoside hydrolase family 66; thermostability