Enzymatic and structural characterization of β-fructofuranosidase from the honeybee gut bacterium Frischella perrara
Enzymatic and structural characterization of β-fructofuranosidase from the honeybee gut bacterium Frischella perrara
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DOI：
10.1007/s00253-022-11863-9
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发表时间：
2022-03-10
影响因子：
5
通讯作者：
Tonozuka, Takashi
中科院分区：
文献类型：
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作者：
Kubota, Arisa;Kawai, Reika;Tonozuka, Takashi
低聚果糖主要是三糖1-蔗果三糖（GF（2））、四糖蔗果三糖（GF（3））和果糖蔗果三糖（GF（4））的混合物。水解GF（3）的酶可用于从低聚果糖混合物制备GF（2）。在大肠杆菌中表达并纯化了来自蜜蜂肠道细菌Frischella perrara（FperFFase）的属于糖苷水解酶家族32（GH 32）的β-呋喃果糖苷酶。分析FperFF酶水解60 mM蔗糖、GF（2）和GF（3）的时间过程，表明FperFF酶对三糖GF（2）的水解活性低于对二糖蔗糖和四糖GF（3）的水解活性。测定了FperFFase的晶体结构及其与果糖复合物的结构。发现FperFF酶与双歧杆菌β-呋喃果糖苷酶在结构上同源，尽管双歧杆菌酶优选水解GF（2），并且在亚位点-1处与果糖相互作用的氨基酸残基在它们之间大部分是保守的。使用定点诱变在Asp 298和Ser 299之间插入脯氨酸残基，并测量变体298 P299的活性。野生型FperFF酶对60 mM GF（2）/GF（3）的活性比率为35.5%，而298 P299的活性比率为23.6%，表明包含Trp 297-Asp 298-Ser 299的环的结构与FperFF酶的底物偏好相关。晶体结构还表明，由残基117-127组成的环可能有助于FperFF酶的底物结合。结果表明，FperFFase在GF（2）的合成中具有潜在的应用价值，Frischella β-果糖呋喃糖苷酶水解蔗果糖的效率高于1-蔗果糖，Trp 297-Asp 298-Ser 299与底物选择性有关，由117-127位残基组成的环有助于底物结合。
Fructooligosaccharide is a mixture of mostly the trisaccharide 1-kestose (GF(2)), tetrasaccharide nystose (GF(3)), and fructosyl nystose (GF(4)). Enzymes that hydrolyze GF(3) may be useful for preparing GF(2) from the fructooligosaccharide mixture. A beta-fructofuranosidase belonging to glycoside hydrolase family 32 (GH32) from the honeybee gut bacterium Frischella perrara (FperFFase) was expressed in Escherichia coli and purified. The time course of the hydrolysis of 60 mM sucrose, GF(2), and GF(3) by FperFFase was analyzed, showing that the hydrolytic activity of FperFFase for trisaccharide GF(2) was lower than those for disaccharide sucrose and tetrasaccharide GF(3). The crystal structure of FperFFase and its structure in complex with fructose were determined. FperFFase was found to be structurally homologous to bifidobacterial beta-fructofuranosidases even though bifidobacterial enzymes preferably hydrolyze GF(2) and the amino acid residues interacting with fructose at subsite - 1 are mostly conserved between them. A proline residue was inserted between Asp298 and Ser299 using site-directed mutagenesis, and the activity of the variant 298P299 was measured. The ratio of activities for 60 mM GF(2)/GF(3) by wild-type FperFFase was 35.5%, while that of 298P299 was 23.6%, indicating that the structure of the loop comprising Trp297-Asp298-Ser299 correlated with the substrate preference of FperFFase. The crystal structure also shows that a loop consisting of residues 117-127 is likely to contribute to the substrate binding of FperFFase. The results obtained herein suggest that FperFFase is potentially useful for the manufacture of GF(2).Key pointsFrischella beta-fructofuranosidase hydrolyzed nystose more efficiently than 1- kestose.Trp297-Asp298-Ser299 was shown to be correlated with the substrate preference.Loop consisting of residues 117-127 appears to contribute to the substrate binding.