Rational conversion of substrate and product specificity in a Salvia monoterpene synthase:: Structural insights into the evolution of terpene synthase function
Rational conversion of substrate and product specificity in a Salvia monoterpene synthase:: Structural insights into the evolution of terpene synthase function
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DOI：
10.1105/tpc.106.047779
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发表时间：
2007-06-01
期刊：
影响因子：
11.6
通讯作者：
Johnson, Christopher B.
中科院分区：
文献类型：
--
作者：
Kampranis, Sotirios C.;Ioannidis, Daphne;Johnson, Christopher B.
萜烯合成酶负责植物和微生物复杂的化学防御库的生物合成。这些似乎都共享一个共同的萜类合成酶折叠的酶，如何确定几乎完全由三种底物中的一种制成的许多不同的产品？对丹参1，8-桉叶油素合成酶(SF-CinS1)结构的阐明，结合对丹参物种内酶的功能和系统发育关系的分析，确定了对产品专一性负责的活性部位残基。因此，SF-CinS1以最少的合理预测的取代成功地转化为莎草烯合成酶，而对水活化所必需的ASN侧链的鉴定将1，8-桉树脑和α-松油醇的活性引入了丹参莎草烯合成酶。SF-CinS1中产物特异性的主要贡献似乎来自形成活性位点的一个螺旋内的局部变形。这种变形在所有其他可用的单萜或倍半萜结构中都可以观察到，这表明了一种保守的机制。此外，单一的氨基酸取代扩大了活性中心的空位，足以容纳更大的法尼基焦磷酸底物，并导致了倍半萜的有效合成，而这一关键氨基酸的交替单一取代产生了额外的5个萜类合成酶。
Terpene synthases are responsible for the biosynthesis of the complex chemical defense arsenal of plants and microorganisms. How do these enzymes, which all appear to share a common terpene synthase fold, specify the many different products made almost entirely from one of only three substrates? Elucidation of the structure of 1,8-cineole synthase from Salvia fruticosa (Sf-CinS1) combined with analysis of functional and phylogenetic relationships of enzymes within Salvia species identified active-site residues responsible for product specificity. Thus, Sf-CinS1 was successfully converted to a sabinene synthase with a minimum number of rationally predicted substitutions, while identification of the Asn side chain essential for water activation introduced 1,8-cineole and alpha-terpineol activity to Salvia pomifera sabinene synthase. A major contribution to product specificity in Sf-CinS1 appears to come from a local deformation within one of the helices forming the active site. This deformation is observed in all other mono- or sesquiterpene structures available, pointing to a conserved mechanism. Moreover, a single amino acid substitution enlarged the active-site cavity enough to accommodate the larger farnesyl pyrophosphate substrate and led to the efficient synthesis of sesquiterpenes, while alternate single substitutions of this critical amino acid yielded five additional terpene synthases.