Abstract
BackgroundRhizopus 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.ResultsTo 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.ConclusionOur 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.
Highlights
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
In AnGA, the highly O-glycosylated linker segment has been shown to be needed for the efficient digestion of raw starch [24,41]
Our present study focuses on functional characterization of the linker region of RoGA
Summary
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. GA under different growth conditions [5], the industrial development of GA has focused only on GA from Aspergillus niger (AnGA; identical to Aspergillus awamori GA) and Rhizopus oryzae (RoGA) because of their stability and high activity [3,6,7]. The overall domain structure of AnGA consists of an N-terminal catalytic region and a C-terminal starch-binding domain (SBD). The organization of that of the RoGA consists of an N-terminal SBD and a C-terminal catalytic region.
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