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


ABSTRACT: 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.

SUBMITTER: Lin SC 

PROVIDER: S-EPMC1933424 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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

Lin Shu-Chuan SC   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  

BMC biochemistry 20070625


<h4>Background</h4>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.<h4>Results</h4>To characterize the linker sequences of RoGA and its involvement in protein expression, a number of RoGA variants containing deletio  ...[more]

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