Enabling microbial syringol conversion through structure-guided protein engineering
Enabling microbial syringol conversion through structure-guided protein engineering
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DOI：
10.1073/pnas.1820001116
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发表时间：
2019-07-09
影响因子：
11.1
通讯作者：
DuBois, Jennifer L.
中科院分区：
文献类型：
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作者：
Machovina, Melodie M.;Mallinson, Sam J. B.;DuBois, Jennifer L.
芳香族化合物的微生物转化是一种新兴的、有前途的植物生物聚合物木质素增值策略。在芳香分解代谢过程中，一个关键且通常限制速率的反应是木质素中丰富的芳香甲氧基的o -芳基去甲基化形成二醇，这使得随后的氧化芳香开环成为可能。最近，一个细胞色素P450系统，GcoAB，被发现去甲基化愈创木酚（2-甲氧基酚），它可以从松柏醇衍生的木质素中产生，形成儿茶酚。然而，天然GcoAB对紫丁香醇（2,6-二甲氧基酚）的去甲基化能力很小，紫丁香醇衍生木质素可以产生类似的化合物。尽管植物中含有丰富的紫丁香醇木质素，但迄今为止尚无紫丁香醇分解代谢途径的报道。在这里，我们使用结构引导蛋白工程使微生物丁香醇与GcoAB的利用。具体来说，苯丙氨酸残基（GcoA-F169）干扰了丁香酚在活性位点的结合，在突变成更小的氨基酸时，实现了有效的丁香酚o -去甲基化。晶体学表明，丁香醇在该变体中采用了有效的结合姿态，分子动力学模拟表明，这是由于消除了GcoA-F169的高柔性侧链与丁香醇的附加甲氧基之间的空间冲突。最后，我们证明了GcoA-F169A变体在恶臭假单胞菌KT2440体内的丁香醛转换。综上所述，我们的研究结果突出了细胞色素P450芳香o -去甲基酶在木质素衍生芳香化合物生物转化中的巨大潜力和可塑性。
Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism is O-aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol. However, native GcoAB has minimal ability to demethylate syringol (2,6-dimethoxyphenol), the analogous compound that can be produced from sinapyl alcohol-derived lignin. Despite the abundance of sinapyl alcohol-based lignin in plants, no pathway for syringol catabolism has been reported to date. Here we used structure-guided protein engineering to enable microbial syringol utilization with GcoAB. Specifically, a phenylalanine residue (GcoA-F169) interferes with the binding of syringol in the active site, and on mutation to smaller amino acids, efficient syringol O-demethylation is achieved. Crystallography indicates that syringol adopts a productive binding pose in the variant, which molecular dynamics simulations trace to the elimination of steric clash between the highly flexible side chain of GcoA-F169 and the additional methoxy group of syringol. Finally, we demonstrate in vivo syringol turnover in Pseudomonas putida KT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromatic O-demethylases in the biological conversion of lignin-derived aromatic compounds.