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Rhizopus oryzae glucoamylase (RoGA) consists of three domains: an amino (N)-terminal …


Biology Articles » Biochemistry » Carbohydrate Biochemistry » Role of the linker region in the expression of Rhizopus oryzae glucoamylase

Abstract
- Role of the linker region in the expression of Rhizopus oryzae glucoamylase

Role of the linker region in the expression of Rhizopus oryzae glucoamylase

Shu-Chuan Lin1, Wei-Ting Liu1, Shi-Hwei Liu1,2, Wei-I Chou1,2, Bor-Kai Hsiung1, I-Ping Lin1, Chia-Chin Sheu2 and Margaret Dah-Tsyr Chang1

1Institute of Molecular and Cellular Biology & Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan 300, Republic of China
2Simpson Biotech Co., Ltd, Taoyuan Country, Taiwan 333, Republic of China

 

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.

Conclusion

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.

BMC Biochemistry 2007, 8:9. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.


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