Reprogramming the chemodiversity of terpenoid cyclization by remolding the active site contour of epi-isozizaene synthase.
Reprogramming the chemodiversity of terpenoid cyclization by remolding the active site contour of epi-isozizaene synthase.
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DOI：
10.1021/bi401643u
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发表时间：
2014-02-25
期刊：
影响因子：
2.9
通讯作者：
Christianson DW
中科院分区：
文献类型：
--
作者：
Li R;Chou WK;Himmelberger JA;Litwin KM;Harris GG;Cane DE;Christianson DW
I类萜类环化酶表异戊二烯合成酶(EIZS)利用普遍存在的非手性异戊二烯底物法尼基二磷酸，产生作为主要倍半萜环化产物的表异戊二烯和至少5个次要倍半萜产物，使EIZS成为探索萜类环化反应中忠诚和混杂的理想平台。EIZS的疏水活性中心轮廓作为模板，强制单一底物构象，而伴侣随后通过明确的机制序列形成碳正离子中间体。在这里，我们以EIZS的晶体结构为指导，系统地改造了26个特定位点突变体文库中的疏水活性位点轮廓。重塑的环化模板不仅通过重新调整野生型酶产生的产物的比例，而且通过产生完全不同结构的新产物来重新编程反应级联。具体地说，我们已经将EIZS产生的特色化产品的总数增加了两倍。此外，我们还将EIZS转化为六种不同的倍半萜合成酶：F96AEIZS是一种(E)-β-法尼烯合成酶，F96WEIZS是一种β-姜黄烯合成酶，F95MEIZS是一种β-二烯合成酶，F198LEIZS是一种β-柏木烯合成酶，F96VEIZS和W203FEIZS是(Z)-γ-双二酚合成酶。活性中心芳香族残基似乎是通过控制临界碳正离子中间体的稳定性和构象来重新编程环化级联反应的热点。大多数突变酶的催化活性只表现出相对温和的2-100倍的损失，这表明负责触发底物电离的残基很容易容忍活性部位腔内更深的突变。
The class I terpenoid cyclase epi-isozizaene synthase (EIZS) utilizes the universal achiral isoprenoid substrate, farnesyl diphosphate, to generate epi-isozizaene as the predominant sesquiterpene cyclization product and at least five minor sesquiterpene products, making EIZS an ideal platform for the exploration of fidelity and promiscuity in a terpenoid cyclization reaction. The hydrophobic active site contour of EIZS serves as a template that enforces a single substrate conformation, and chaperones subsequently formed carbocation intermediates through a well-defined mechanistic sequence. Here, we have used the crystal structure of EIZS as a guide to systematically remold the hydrophobic active site contour in a library of 26 site-specific mutants. Remolded cyclization templates reprogram the reaction cascade not only by reproportioning products generated by the wild-type enzyme but also by generating completely new products of diverse structure. Specifically, we have tripled the overall number of characterized products generated by EIZS. Moreover, we have converted EIZS into six different sesquiterpene synthases: F96A EIZS is an (E)-β-farnesene synthase, F96W EIZS is a zizaene synthase, F95H EIZS is a β-curcumene synthase, F95M EIZS is a β-acoradiene synthase, F198L EIZS is a β-cedrene synthase, and F96V EIZS and W203F EIZS are (Z)-γ-bisabolene synthases. Active site aromatic residues appear to be hot spots for reprogramming the cyclization cascade by manipulating the stability and conformation of critical carbocation intermediates. A majority of mutant enzymes exhibit only relatively modest 2–100-fold losses of catalytic activity, suggesting that residues responsible for triggering substrate ionization readily tolerate mutations deeper in the active site cavity.
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DOI：
10.1351/pac-con-09-09-37
发表时间：
2010
期刊：
Pure and applied chemistry. Chimie pure et appliquee
影响因子：
--
作者：
Aaron JA;Christianson DW
通讯作者：
Christianson DW
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
3.6
作者：
CANE, DE;YANG, GH;HOHN, TM
通讯作者：
HOHN, TM
DOI：
10.1073/pnas.0601605103
发表时间：
2006-06-27
影响因子：
11.1
作者：
Greenhagen, Bryan T.;O'Maille, Paul E.;Chappell, Joe
通讯作者：
Chappell, Joe
影响因子：
8.4
作者：
Anthony, Jennifer R.;Anthony, Larry C.;Keasling, Jay D.
通讯作者：
Keasling, Jay D.