title: Novel dextranase catalyzing cycloisomaltooligosaccharide formation and identification of catalytic amino acids and their functions using chemical rescue approach.
abstract: A novel endodextranase from Paenibacillus sp. (Paenibacillus sp. dextranase; PsDex) was found to mainly produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides (CIs) with a degree of polymerization of 7-14 from dextran. The 1,696-amino acid sequence belonging to the glycosyl hydrolase family 66 (GH-66) has a long insertion (632 residues; Thr(451)-Val(1082)), a portion of which shares identity (35% at Ala(39)-Ser(1304) of PsDex) with Pro(32)-Ala(755) of CI glucanotransferase (CITase), a GH-66 enzyme that catalyzes the formation of CIs from dextran. This homologous sequence (Val(837)-Met(932) for PsDex and Tyr(404)-Tyr(492) for CITase), similar to carbohydrate-binding module 35, was not found in other endodextranases (Dexs) devoid of CITase activity. These results support the classification of GH-66 enzymes into three types: (i) Dex showing only dextranolytic activity, (ii) Dex catalyzing hydrolysis with low cyclization activity, and (iii) CITase showing CI-forming activity with low dextranolytic activity. The fact that a C-terminal truncated enzyme (having Ala(39)-Ser(1304)) has 50% wild-type PsDex activity indicates that the C-terminal 392 residues are not involved in hydrolysis. GH-66 enzymes possess four conserved acidic residues (Asp(189), Asp(340), Glu(412), and Asp(1254) of PsDex) of catalytic candidates. Their amide mutants decreased activity (1⁄1,500 to 1⁄40,000 times), and D1254N had 36% activity. A chemical rescue approach was applied to D189A, D340G, and E412Q using α-isomaltotetraosyl fluoride with NaN(3). D340G or E412Q formed a β- or α-isomaltotetraosyl azide, respectively, strongly indicating Asp(340) and Glu(412) as a nucleophile and acid/base catalyst, respectively. Interestingly, D189A synthesized small sized dextran from α-isomaltotetraosyl fluoride in the presence of NaN(3).
authors.affiliation: Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.
authors.forename: Young-Min
authors.initials: YM
authors.lastname: Kim
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Kiso
authors.forename: Tomoe
authors.initials: T
authors.lastname: Muraki
authors.forename: Min-Sun
authors.initials: MS
authors.lastname: Kang
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakai
authors.forename: Wataru
authors.initials: W
authors.lastname: Saburi
authors.forename: Weeranuch
authors.initials: W
authors.lastname: Lang
authors.forename: Hee-Kwon
authors.initials: HK
authors.lastname: Kang
authors.forename: Masayuki
authors.initials: M
authors.lastname: Okuyama
authors.forename: Haruhide
authors.initials: H
authors.lastname: Mori
authors.forename: Ryuichiro
authors.initials: R
authors.lastname: Suzuki
authors.forename: Kazumi
authors.initials: K
authors.lastname: Funane
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Suzuki
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Momma
authors.forename: Zui
authors.initials: Z
authors.lastname: Fujimoto
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Oguma
authors.forename: Mikihiko
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Doman
authors.initials: D
authors.lastname: Kim
authors.forename: Atsuo
authors.initials: A
authors.lastname: Kimura
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D003910:Dextranase / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism; D003911:Dextrans / Q000737:chemistry* / Q000378:metabolism; D020125:Mutation, Missense; D056507:Paenibacillus / Q000201:enzymology* / Q000235:genetics; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D003911:Dextrans; D003910:Dextranase