Design of a Superpositively Charged Enzyme: Human Carbonic Anhydrase II Variant with Ferritin Encapsulation and Immobilization
Design of a Superpositively Charged Enzyme: Human Carbonic Anhydrase II Variant with Ferritin Encapsulation and Immobilization
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DOI：
10.1021/acs.biochem.1c00515
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发表时间：
2021-11-10
期刊：
影响因子：
2.9
通讯作者：
Dmochowski, Ivan J.
中科院分区：
文献类型：
--
作者：
Bulos, Joshua A.;Guo, Rui;Dmochowski, Ivan J.
超荷电蛋白质表现出高溶解度和其他理想的性质，但以前没有工程改造的超正电荷酶。蛋白质如绿色荧光蛋白的带超级电荷的变体已经被有效地封装在闪烁古生球菌嗜热铁蛋白（AfFtn）内。通过超分子铁蛋白的包封可以产生具有多种螯合货物的系统。为了推进在酶学和绿色化学中的应用，我们寻求一种用于增压酶的通用方法，该酶保留活性并且与AfFtn包封相容。锌金属酶人碳酸酐酶II（hCAII）是一个有吸引力的封装目标，基于其水解活性和生理转化的二氧化碳碳酸氢盐。计算设计的hCAII变体在蛋白质表面的19个位点上含有带正电荷的残基，导致推定的净电荷从-1变为+21。这种设计的hCAII（+21）表现出在AfFtn内的包封，而不需要融合配偶体或额外的试剂。hCAII（+21）变体保留了与野生型相当的酯酶活性，并自发地在其周围模板化AfFtn 24聚体的组装。与hCAII（+21）相比，AfFtn-hCAII（+21）主体-客体复合物表现出更高的活性和热稳定性。在固定在固体支持物上后，AfFtn-hCAII（+21）保留酶活性并在升高的温度下表现出活性增强。
Supercharged proteins exhibit high solubility and other desirable properties, but no engineered superpositively charged enzymes have previously been made. Superpositively charged variants of proteins such as green fluorescent protein have been efficiently encapsulated within Archaeoglobus fulgidus thermophilic ferritin (AfFtn). Encapsulation by supramolecular ferritin can yield systems with a variety of sequestered cargo. To advance applications in enzymology and green chemistry, we sought a general method for supercharging an enzyme that retains activity and is compatible with AfFtn encapsulation. The zinc metalloenzyme human carbonic anhydrase II (hCAII) is an attractive encapsulation target based on its hydrolytic activity and physiologic conversion of carbon dioxide to bicarbonate. A computationally designed variant of hCAII contains positively charged residues substituted at 19 sites on the protein's surface, resulting in a shift of the putative net charge from -1 to +21. This designed hCAII(+21) exhibits encapsulation within AfFtn without the need for fusion partners or additional reagents. The hCAII(+21) variant retains esterase activity comparable to the wild type and spontaneously templates the assembly of AfFtn 24mers around itself. The AfFtn-hCAII(+21) host-guest complex exhibits both greater activity and thermal stability when compared to hCAII(+21). Upon immobilization on a solid support, AfFtn-hCAII(+21) retains enzymatic activity and exhibits an enhancement of activity at elevated temperatures.