Engineering a Monolignol 4-O-Methyltransferase with High Selectivity for the Condensed Lignin Precursor Coniferyl Alcohol
Engineering a Monolignol 4-O-Methyltransferase with High Selectivity for the Condensed Lignin Precursor Coniferyl Alcohol
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DOI：
10.1074/jbc.m115.684217
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发表时间：
2015-10-30
影响因子：
4.8
通讯作者：
Liu, Chang-Jun
中科院分区：
文献类型：
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作者：
Cai, Yuanheng;Bhuiya, Mohammad-Wadud;Liu, Chang-Jun
木质素是植物细胞壁中的一种刚性生物聚合物，源自三种木质素单体的氧化聚合。木质素单体的组成决定了木质素缩合的程度、反应性，从而决定了植物细胞壁的可降解性。愈创木基木质素被视为缩合结构单元。木质素的聚合是通过木质素单体的对羟基的去质子化引发的。因此，优先修饰特定木质素单体的对位羟基以剥夺其脱氢倾向将干扰特定木质素亚基的形成。在这里，我们测试了这样一个假设：对木质醇单体 4-O-甲基转移酶的活性位点进行特异性重塑将产生一种能够特异性甲基化缩合愈创木基木质素前体松柏醇的酶。将晶体结构信息与组合活性位点饱和诱变相结合，并从工程混杂酶 MOMT5（T133L/E165I/F175I/F166W/H169F）开始，通过添加四个取代（即 M26H、S30R、V33S 和 T319M）逐步重塑其底物结合口袋， 产生即使在过量芥子醇存在下也能够有区别地醚化松柏醇的对羟基的突变酶。工程酶变体具有显着减少的底物结合袋，其施加明显的空间位阻，从而排除体积较大的木质素前体。所得酶变体代表了调节植物中木质素组成和/或结构的极好候选者。
Lignin, a rigid biopolymer in plant cell walls, is derived from the oxidative polymerization of three monolignols. The composition of monolignol monomers dictates the degree of lignin condensation, reactivity, and thus the degradability of plant cell walls. Guaiacyl lignin is regarded as the condensed structural unit. Polymerization of lignin is initiated through the deprotonation of the para-hydroxyl group of monolignols. Therefore, preferentially modifying the para-hydroxyl of a specific monolignol to deprive its dehydrogenation propensity would disturb the formation of particular lignin subunits. Here, we test the hypothesis that specific remodeling the active site of a monolignol 4-O-methyltransferase would create an enzyme that specifically methylates the condensed guaiacyl lignin precursor coniferyl alcohol. Combining crystal structural information with combinatorial active site saturation mutagenesis and starting with the engineered promiscuous enzyme, MOMT5(T133L/E165I/F175I/F166W/H169F), we incrementally remodeled its substrate binding pocket by the addition of four substitutions, i.e. M26H, S30R, V33S, and T319M, yielding a mutant enzyme capable of discriminately etherifying the para-hydroxyl of coniferyl alcohol even in the presence of excess sinapyl alcohol. The engineered enzyme variant has a substantially reduced substrate binding pocket that imposes a clear steric hindrance thereby excluding bulkier lignin precursors. The resulting enzyme variant represents an excellent candidate for modulating lignin composition and/or structure in planta.