A novel domain arrangement in a monomeric cyclodextrin-hydrolyzing enzyme from the hyperthermophile Pyrococcus furiosus.
PFTA (Pyrococcus furiosus thermostable amylase) is a hyperthermophilic amylase isolated from the archaeon Pyrococcus furiosus. This enzyme possesses characteristics of both α-amylase- and cyclodextrin (CD)-hydrolyzing enzymes, allowing it to degrade pullulan, CD and acarbose-activities that are absent in most α-amylases-without the transferring activity that is common in CD-hydrolyzing enzymes. The crystal structure of PFTA revealed a unique monomeric subunit with an extended N-terminal region and an N'-domain folded into its own active site-a significantly altered domain configuration relative to that of the conventional dimeric CD-hydrolyzing amylases in glycoside hydrolase family 13. The active site is formed by the interface of the N'-domain and the catalytic domain and exhibits a broad and wide-open geometry without the concave pocket that is commonly found in the active sites of maltogenic amylases. The mutation of a residue (Gly415 to Glu) located at the domain interface between the N'- and catalytic domains yielded an enzyme that produced a significantly higher purity maltoheptaose (G7) from β-CD, supporting the involvement of this interface in substrate recognition and indicating that this mutant enzyme is a suitable candidate for the production of pure G7. The unique configuration of the active site distinguishes this archaic monomeric enzyme from classical bacterial CD-hydrolyzing amylases and provides a molecular basis for its enzymatic characteristics and for its potential use in industrial applications.
Department of Food Science and Technology, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 305-764, Republic of Korea.
Jong-Tae
JT
Park
Hyung-Nam
HN
Song
Tae-Yang
TY
Jung
Myoung-Hee
MH
Lee
Sung-Goo
SG
Park
Eui-Jeon
EJ
Woo
Kwan-Hwa
KH
Park
D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D005959:Glucosidases / Q000737:chemistry* / Q000235:genetics; D020125:Mutation, Missense; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D000516:alpha-Amylases / Q000737:chemistry* / Q000235:genetics
D019843:Archaeal Proteins; D005959:Glucosidases; C030597:cyclodextrin glucoamylase; D000516:alpha-Amylases