Department of Chemistry, Marquette University, PO Box 1881, Milwaukee, WI 53201-1881, United States; Department of Chemistry and Biochemistry, Loyola University Chicago, 1068 W Sheridan Rd., Chicago, IL 60660, United States.
Department of Chemistry, Marquette University, PO Box 1881, Milwaukee, WI 53201-1881, United States.
Biochim Biophys Acta Proteins Proteom. 2017 Jan;1865(1):107-112. doi: 10.1016/j.bbapap.2016.09.013. Epub 2016 Sep 28.
Nitrile hydratase (NHase), an industrially important enzyme that catalyzes the hydration of nitriles to their corresponding amides, has only been characterized from prokaryotic microbes. The putative NHase from the eukaryotic unicellular choanoflagellate organism Monosiga brevicollis (MbNHase) was heterologously expressed in Escherichia coli. The resulting enzyme expressed as a single polypeptide with fused α- and β-subunits linked by a seventeen-histidine region. Size-exclusion chromatography indicated that MbNHase exists primarily as an (αβ) homodimer in solution, analogous to the αβ homotetramer architecture observed for prokaryotic NHases. The NHase enzyme contained its full complement of Co(III) and was fully functional without the co-expression of an activator protein or E. coli GroES/EL molecular chaperones. The homology model of MbNHase was developed identifying Cys400, Cys403, and Cys405 as active site ligands. The results presented here provide the first experimental data for a mature and active eukaryotic NHase with fused subunits. Since this new member of the NHase family is expressed from a single gene without the requirement of an activator protein, it represents an alternative biocatalyst for industrial syntheses of important amide compounds.
腈水合酶（NHase）是一种重要的工业酶，能够催化腈转化为相应的酰胺。该酶仅在原核微生物中得到了描述。从单细胞真核纤毛原生动物 Monosiga brevicollis（MbNHase）中分离出的假定 NHase 在大肠杆菌中异源表达。表达的酶为融合的 α-和 β-亚基的单一条多肽，通过十七个组氨酸区域连接。分子筛层析表明，MbNHase 主要以溶液中的（αβ）同源二聚体形式存在，类似于观察到的原核 NHase 的αβ四聚体结构。该 NHase 酶含有完整的 Co(III)配位，无需表达激活蛋白或大肠杆菌 GroES/EL 分子伴侣即可完全发挥功能。MbNHase 的同源模型确定了 Cys400、Cys403 和 Cys405 为活性位点配体。本研究提供了第一个关于成熟且具有融合亚基的真核 NHase 的实验数据。由于这个 NHase 家族的新成员由单个基因表达，不需要激活蛋白，因此它代表了用于工业合成重要酰胺化合物的替代生物催化剂。
