米根黴菌 (Rhizopus oryzae) 葡萄糖水解酶 (glucoamylase) 的整體結構包含胺基端的澱粉吸附區 (starch binding domain、RoSBD)、羧基端的催化區及醣基化的連接片段。RoSBD隸屬於醣類吸附模組 (carbohydrate-binding module、 CBM) 家族二十一，能與顆粒狀生澱粉及可溶性多醣結合。核磁共振與結晶繞射解析之RoSBD三級結構證明其具有兩個配體結合位，分別由色胺酸47、酪胺酸83、酪胺酸93、及酪胺酸94組成第一個結合位；由酪胺酸32、苯丙胺酸58、及酪胺酸67組成第二個結合位。RoSBD與配體結合主要依靠芳香環胺基酸與醣類六角環形成疏水性交互作用。本研究首先以恆溫滴定熱量計 (ITC)，螢光光譜，及顆粒澱粉結合飽和曲線分析 (depletion isotherm) 測量不同醣類與RoSBD結合的親合力，區分各種構形、長度、醣苷鍵組成的醣類與RoSBD親合力的差別。進一步以單點突變探討RoSBD兩個配體結合位對於可溶性醣類的結合力強弱，發現第二個結合位的酪胺酸32突變後，RoSBD與水溶性配體的親合力顯著地減弱。搭配結晶繞射結果發現RoSBD與醣類結合後，酪胺酸32的支鏈產生明顯位移翻轉，並與苯丙胺酸58形成一對結合鉗 (binding clamp) 共同穩定結合配體，證明酪胺酸32在RoSBD與可溶性醣類結合過程中扮演重要的角色。另外富含天門冬胺酸的兩段環 (polyN loop)結構亦會形成結合鉗，提供氫鍵以結合配體。本實驗室先前的研究已證明第一個結合位的色胺酸47主要負責與不可溶性醣類結合，因此本研究推論RoSBD兩個配體結合位扮演不同的醣類結合角色：第一個結合位中色胺酸47與富含天門冬胺酸的環會先和澱粉結合並促使澱粉螺旋狀結構鬆散，暴露出更多水溶性表面積，再由第二個結合位的酪胺酸32與苯丙胺酸58形成結合鉗，結合此水溶性澱粉表面使之更形鬆散以利酵素催化水解。本研究結合生物資訊分析，驗證RoSBD與配體結合的特性、兩個結合位角色及芳香環胺基酸的保留性，進一步統整SBD及CBM結構與功能之相關性。

Rhizopus oryzae glucoamylase is composed of an N-terminal starch binding domain (RoSBD, residues 26-131) and a C-terminal catalytic domain (residues 168-604) connected by an O-glycosylated linker (residues 132-167). RoSBD is categorized to carbohydrate binding modules (CBMs) family 21, and has been demonstrated to effectively adsorb onto raw starch and other soluble oligosaccharides. Three dimensional structures of RoSBD in the presence of a cyclic ligand β-cyclodextrin (βCD) or a linear maltoheptaose (G7) have been determined by NMR and X-ray crystallography. RoSBD possesses two ligand binding sites, residues Trp47, Tyr83, Tyr93, and Tyr94 constitute binding site I, and residues Tyr32, Phe58, and Tyr67 form binding site II. Hydrophobic interaction between the sugar rings of ligands and aromatic residues of RoSBD act as a key determinant of overall binding affinity and specificity. Here isothermal titration calorimetry (ITC), fluorescence spectrophotometry and depletion isotherm have been used to identify binding affinities of various glycans containing cyclic and linear linkages with special focus on the deterministic factors such as length, glycosidic linkage, ring size, and solubility of polysaccharides. Micromolar range binding affinities of different ligands are observed. In addition, to distinguish the affinity of two ligand binding sites, key binding residue of two sites, Trp47 in site I and Tyr32, Phe58 in site II are specifically mutated. Interestingly, Tyr32 mutant (Y32A) shows over 10 fold weaker binding affinity than wild-type RoSBD, strongly suggesting that Tyr32 plays a more important role in binding soluble glycans. X-ray crystallography results indicate that the side chain of Tyr32 significantly flips over to form a ligand binding clamp with Phe58 upon binding to either βCD or G7. In addition, unique polyN loops in binding site I form another ligand binding clamp to cooperatively interact with ligand through hydrogen bonding. The two binding sites of RoSBD appear to play distinct roles in ligand binding. Trp47 along with polyN loops initially bind to insoluble starch strands and force starch to twist apart to expose extensive surface for solvent access. In addition, Tyr32 and Phe58 in binding site II form a ligand binding clamp to coordinately bind to soluble starch with high binding affinity. Finally, bioinformatic analysis demonstrates binding properties, correlation of two ligand binding sites, and conservation of binding residues of RoSBD, which in turn leads to more understanding in structure-function relationship of SBD and CBM.

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