Role of the linker region in the expression of Rhizopus oryzae glucoamylase.
BACKGROUND
Rhizopus oryzae glucoamylase (RoGA) consists of three domains: an amino (N)-terminal raw starch-binding domain (SBD), a glycosylated linker domain, and a carboxy (C)-terminal catalytic domain. The 36-amino-acid linker region (residues 132-167) connects the two functional domains, but its structural and functional roles are unclear.


RESULTS
To characterize the linker sequences of RoGA and its involvement in protein expression, a number of RoGA variants containing deletions and mutations were constructed and expressed in Saccharomyces cerevisiae. Deletion analyses demonstrate that the linker region, especially within residues 161 to 167, is required for protein expression. In addition, site-directed mutagenesis and deglycosylation studies reveal that the linker region of RoGA contains both N- and O-linked carbohydrate moieties, and the N-linked oligosaccharides play a major role in the formation of active enzyme. Although the linker segment itself appears to have no ordered secondary structural conformation, the flexible region indeed contributes to the stabilization of functional N- and C-terminal domains.


CONCLUSIONS
Our data provide direct evidence that the length, composition, and glycosylation of the interdomain linker play a central role in the structure and function of RoGA.
Lin
Shu-Chuan
SC
Institute of Molecular and Cellular Biology & Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China. d938213@oz.nthu.edu.tw <d938213@oz.nthu.edu.tw>
Liu
Wei-Ting
WT
Liu
Shi-Hwei
SH
Chou
Wei-I
WI
Hsiung
Bor-Kai
BK
Lin
I-Ping
IP
Sheu
Chia-Chin
CC
Dah-Tsyr Chang
Margaret
M
D000595:Amino Acid Sequence; D015966:Gene Expression Regulation, Fungal / Q000502:physiology*; D005087:Glucan 1,4-alpha-Glucosidase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D012233:Rhizopus / Q000201:enzymology* / Q000235:genetics*
D010446:Peptide Fragments; D005087:Glucan 1,4-alpha-Glucosidase