Morehouse Benjamin R, Kumar Ramasamy P, Matos Jason O, Olsen Sarah Naomi, Entova Sonya, Oprian Daniel D
Department of Biochemistry, Brandeis University , Waltham, Massachusetts 02454, United States.
Biochemistry. 2017 Mar 28;56(12):1706-1715. doi: 10.1021/acs.biochem.7b00143. Epub 2017 Mar 15.
Terpenes make up the largest and most diverse class of natural compounds and have important commercial and medical applications. Limonene is a cyclic monoterpene (C) present in nature as two enantiomers, (+) and (-), which are produced by different enzymes. The mechanism of production of the (-)-enantiomer has been studied in great detail, but to understand how enantiomeric selectivity is achieved in this class of enzymes, it is important to develop a thorough biochemical description of enzymes that generate (+)-limonene, as well. Here we report the first cloning and biochemical characterization of a (+)-limonene synthase from navel orange (Citrus sinensis). The enzyme obeys classical Michaelis-Menten kinetics and produces exclusively the (+)-enantiomer. We have determined the crystal structure of the apoprotein in an "open" conformation at 2.3 Å resolution. Comparison with the structure of (-)-limonene synthase (Mentha spicata), which is representative of a fully closed conformation (Protein Data Bank entry 2ONG ), reveals that the short H-α1 helix moves nearly 5 Å inward upon substrate binding, and a conserved Tyr flips to point its hydroxyl group into the active site.
萜类化合物是天然化合物中最大且最多样化的一类，具有重要的商业和医学应用。柠檬烯是一种环状单萜（C），在自然界中以两种对映体（+）和（-）的形式存在，它们由不同的酶产生。（-）-对映体的产生机制已得到详细研究，但要了解这类酶如何实现对映体选择性，同样重要的是要对生成（+）-柠檬烯的酶进行全面的生化描述。在此，我们报告了从脐橙（Citrus sinensis）中首次克隆并对（+）-柠檬烯合酶进行生化特性分析。该酶遵循经典的米氏动力学，且仅产生（+）-对映体。我们已确定脱辅基蛋白处于“开放”构象时2.3 Å分辨率的晶体结构。与代表完全封闭构象的（-）-柠檬烯合酶（留兰香薄荷（Mentha spicata））的结构（蛋白质数据库条目2ONG）进行比较，结果表明，短的H-α1螺旋在底物结合时向内移动近5 Å，并且一个保守的酪氨酸翻转，使其羟基指向活性位点。
