Many raw starch-degrading enzymes have a modular structure in which the functional domains are spatially separated by linker sequences. In AnGA, the highly O-glycosylated linker segment has been shown to be needed for the efficient digestion of raw starch [24,41]. In contrast, the raw starch-digesting α-amylase from the yeast Cryptococcus sp. strain S-2, lacking of a linker segment demonstrates that the linker region is not always essential for the discrete domains in raw starch-degrading enzymes to retain their proper function . Our present study focuses on functional characterization of the linker region of RoGA. The linker between the SBD and the catalytic domain consists of 36 amino acids with several oligosaccharides attached, and the region facilitates the formation of functional RoGA in S. cerevisiae. The GAΔ132–167 deletion mutant failed to produce detectable protein at 30°C, presumably because the distance between the two domains was too short for them to be folded and secreted correctly. Interestingly, prolonged incubation at low temperature was allowed to synthesize the mutant, which showed detectable activity as visualized on starch plate (Figure 4C No. 3), indicating that low temperature might work in concert to increase the de novo synthesis and stabilize correct folding of the mutant protein. In addition, our results show that the properties of two functional domains are influenced by the linker region. The linker sequence influences the substrate binding process, possibly in the substrate transfer step. Analysis of the isolated catalytic domain shows that the formation of functional domain requires the linker region, especially the amino acid sequences from 161 to 167. Interestingly, the single N-glycosylation site within the linker domain, Asn167, is demonstrated to be extremely important for the function of RoGA. Some glycoproteins need the N-glycans during synthesis for proper folding, sufficient for the efficient secretion or increased stability [43-45]. In this report, we have demonstrated that the N-linked oligomannosides in the linker region play an important role in expression of functional RoGA, however, the exact structures of the oligosaccharides attached to the linker segment remain to be further investigated.
Prior to our present work, no three dimensional structure of intact RoGA has ever been reported. The catalytic domain of RoGA shows 36% identity and 52% similarity to that of AnGA, a functional domain with a characteristic α-helical structure. As for the three dimensional structure of SBD, it has been initially molecular modeled by structural bioinformatics methodology  and subsequently determined by NMR spectroscopy . Interestingly, while the SBDs of RoGA and AnGA share extremely low similarity in their primary structures, their secondary and tertiary structures appear to be quite similar, strongly suggesting that SBDs from CBM20 and CBM21 can be grouped into a new clan with similar functional structures [27,28]. It has been clearly demonstrated that the presence of the linker region leads to much more stable conformation of the two discrete functional domains. Taken together, our data provide several lines of direct evidence that the linker region of RoGA plays a crucial role in terms of holding structural integrity, enhancing structure stability, and facilitating functional protein expression.