生物信息学筛选催化茶黄素合成的多酚氧化酶

Bioinformatic Screening of Natural Polyphenol Oxidases for Theaflavins Synthesis

利用生物信息学方法从天然植物中筛选天然PPO酶源.用PPO保守序列从Uniport中检索到529条植物源PPO,除去重复的和不完整的序列后,得到178条PPO序列.用同源建模法,以甘薯PPO晶体结构(1BUG)为模板,构建各PPO的三维结构模型,经Procheck和Verify3D评价后,获得163个优质的PPO三维结构.用TM-score(空间拓扑)和RMSD(结构叠合)对163个PPO与茶PPO(A6N8J4)的三维结构进行了相似性比较.用Autodock Vina将163个PPO与茶中4种主要儿茶素进行分子对接,根据酶与底物结合的亲和力(Binding Affinity)预测各PPO的活性,发现酶与底物的亲和力与酶的一级结构序列同源性和酶的三维结构的重叠度之间无直接关系.从潜在的10种高PPO活性的植物材料中提取PPO、测定其茶黄素转化活性.10种植物材料中均有不同程度的PPO活性,其中沙梨和甘薯的PPO活性最高,且高于茶叶PPO活性.甘薯资源极其丰富,可从甘薯生产淀粉的废水中大量回收PPO,用于茶黄素的工业合成.本研究表明,利用生物信息学法可以从大量酶的序列信息中成功筛选出高活性的酶,与传统的酶活性筛选法比,可大大提高筛选范围和效率.

We developed a bioinformatic approach to screen natural active PPOs. Using the PPO core sequence （ pfam PF00264）, 529 plant PPOs were blasted in the Uniprot database. After deducted incomplete, synthetic and repeated sequences, 178 independent plant PPOs with higher than 30% identity to tea PPO （A6N8J4） were obtained. The 3-D structures of 163 PPOs were constructed based on the PPO crystal structure of sweet potato （PDB 1BUG） , optimized by molprobity and evaluated using the Procheck and Verify3D programs. The similarities of spatial structures of 163 PPO models were compared to tea PPO using the TM-score and RMSD systems. The 163 PPOs were docked with tea catechins as ligands using Autodock-Vina programs, and their potential enzyme activities were quantitatively evaluated by their binding affinity. Analysis indicated that PPO activity may mainly rely on some specific amino acids at the reaction center, but not on its homology of amino acids and spatial structure. The enzyme activities of 10 top potential plants were analyzed. The results showed that Chinese pear and sweet potato had high PPO activity, higher than tea PPO. The rich resource of sweet potato allows large production of the en- zyme for industrial application. This study indicated the bioinformatic screening is an efficient method in finding out highly active enzymes from a large number of sequences.