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[学术文献 ] Identification of Subfamily Specific Residues within Highly Active and Promiscuous Alcohol Dehydrogenases 进入全文
ACS Catalysis
Enzyme selection is an essential process in the biobased production of chemicals. It is essential to develop a method to extract yet unknown useful enzymes from protein databases. Enzymes that exhibit substrate promiscuity and high activity hold the potential to access unknown reactions and mediate known reactions with a higher performance. Herein, we propose and validate a principal component analysis (PCA)-based classification method, termed MUSASHI (MUltiple-Sequence Alignment-based protein Selection via clustering using HIgh-dimensional analysis), to identify subfamily-specific residues that are highly conserved among promiscuous alcohol dehydrogenase (ADH). Specifically, zinc-dependent ADH homologues retrieved from the protein database were classified into 9 groups, and according to PCA-based clustering, the activities of 18 ADHs, with representative enzymes from each group, were characterized. As a result, we identified two promiscuous ADH groups: Group 1 ADH, efficient with short-chain and aromatic aldehydes, and Group 3 ADH, efficient with aliphatic and aromatic ketones. Sequence feature analysis then revealed subfamily-specific residues, which are highly conserved only in promiscuous ADH Groups 1 and 3, with the potential to biosynthesize a wide spectrum of target compounds. Tatumella ptyseos ADH, identified from Group 1 of this study, showed higher isobutanol and 2-phenylethanol bioconversions than that of a conventional ADH (Ahr). These results indicate that the MUSASHI method for subfamily-specific residue identification can enable optimal enzyme selection from protein databases.
[前沿资讯 ] 光酶催化官能团远端手性中心的对映汇聚 进入全文
科学网
2025年6月24日，美国伊利诺伊大学香槟分校（University of Illinois Urbana-Champaign）赵惠民教授团队在Nature Catalysis期刊在线发表了题为“Photoenzymatic stereoablative enantioconvergence of γ-chiral oximes via hydrogen atom transfer”的研究成果。 该研究报道了一种基于光酶催化氢原子转移的对映汇聚策略，实现官能团远端的γ-手性碳氢键的立体选择性重构。由外消旋体合成对映纯的手性分子在药物、材料和天然产物合成中有广泛的应用前景。尽管传统的有机合成方法在官能团近端手性构建方面已取得显著进展，但对于远离官能团的碳氢键实现高效、对映选择性的构筑，仍是当前化学方法的难题。主要限制因素是官能团远端碳氢键的活性较低、空间位阻难以控制，以及存在复杂的副反应。相比之下，生物催化具备独特优势。酶以其高度结构化的活性位点，能够在温和条件下实现对底物的精确识别与空间调控，尤其在立体选择性转化中表现出色。近年来，借助定向进化与人工设计，许多天然的非光酶被成功“改造”以参与光诱导自由基反应，拓展了酶在非天然反应中的应用边界。 在该项研究中，赵惠民团队设计了一种结合光诱导单电子转移与氢原子转移（HAT）的催化体系，利用黄素依赖型烯还原酶（ERED）在蓝光照射下形成亚胺基自由基，诱发γ-位点的1,5-HAT实现手性信息的“消除”，随后通过酶活性位点控制的对映选择性HAT完成产物的手性重建，最终通过水解生成γ-手性酮产物。该策略突破了传统酶催化的空间限制，在实现反应远距立体控制的同时，避免了半量原料损耗，理论上可实现100%的对映体转化率。从机制上看，作者通过一系列实验和计算发现：R/S底物在该体系中具有相似的反应性；γ-手性中心的氢原子主要来源于还原态黄素（FMNH-）；存在亚胺基自由基直接从半醌态黄素（FMNsq）获得氢的副反应，和目标1,5-HAT构成竞争。 在此基础上，研究团队进一步引入多轮对映汇聚催化循环，有效富集优势构型产物，最高可实现最高97:3 e.r.与99%的产率。该方法适用于一系列含芳香基或杂环结构的γ-手性底物，展现出一定的底物适应性。该策略有望应用于药物合成、材料功能化及分子骨架改造等前沿领域，为发展绿色、精准的生物合成技术提供新的思路。
[学术文献 ] Enhancing the thermal stability and activity of the engineered self-sufficient P450SPα-SOX by switching the domains linker 进入全文
International Journal of Biological Macromolecules
This work reports on the engineering of the linker between P450 SPα (CYP152B1) and sarcosine oxidase (SOX), with the aim of enhancing the structural rigidity of the fusion protein (SPα-SOX) and study the effect on its stability and catalytic performance. Differential scanning calorimetry shows that the construct bearing the rigid linker (SPα-rigidSOX) results in a higher energy barrier to unfolding (765 kcal/mol) compared to the previous fusion system (SPα-flexible-SOX) (561 kcal/mol), as well as a Tonset above 50 °C. Furthermore, residual CO-binding after heat treatment was investigated for both the fusion systems, and a 5.7 °C increase of the T50 of SPα-rigid-SOX is shown. Interestingly, a stabilized P420 semifolded state of the SPα is also observed after SPα-rigid-SOX incubation at high temperature (40°). The two fusion systems were studied at high temperature for the turnover of lauric acid: SPα-rigid-SOX shows a 98 % conversion yield using 5 mM substrate compared to the 24 % conversion of SPαflexibleSOX when the catalysis is carried out at 40 °C. Finally, the activity of the two constructs was tested using styrene as a substrate, and three products of catalysis were observed: styrene oxide (85 %), phenylacetaldehyde (0–3 %) and 2-phenylpropenal (12–15 %). Interestingly, 2-phenylpropenal is observed for the first time and only for the fusion enzymes. Also in this case, SPα-rigid-SOX outperformed SPα-flexible-SOX with a 3-fold higher conversion yield. Overall, we demonstrate that the rigid linker improves the fusion enzyme thermal stability and catalytic performance, both at high temperature and in mild conditions, resulting also in the production of new molecules of biotechnological interest.
[学术文献 ] Discovery to Engineering of Mycotoxin Deoxynivalenol Degrading Enzymes Based on the Specialized Glyoxalase I 进入全文
Advanced Science
Deoxynivalenol (DON) is a mycotoxin that is omnipresent in food and feed. Therefore, this study has focused on discovery, molecular characterization, and engineering of DON degrading enzymes, based on a DON isomerizing enzyme (e.g., the specialized glyoxalase I from Gossypium raimondii (Gr-SPG)). A molecular phylogeny-based sequence and structure analysis elucidated the evolutionary trajectory of the DON degrading enzymes. Ancestral sequence reconstruction led to the generation of thermostable evolutionary intermediates of SPG (e.g., Anc216). Molecular modeling and consensus protein design allowed to understand the structure and function relationships and also identify the key conserved mutations that influence catalytic activity and thermostability. Ultimately, a highly active and thermostable SPG (e.g., a quintuple mutant of Anc216 (Anc216_M5)) was constructed from a newly discovered extant SPG enzyme (OR9). The Anc216_M5 exhibited a T5010 of 68 °C, which is 16.3 °C higher than that of the wild-type enzyme. Furthermore, the engineered enzyme showed 40% greater DON degrading activity than OR9, which is significantly higher than that of Gr-SPG. Therefore, it is assumed that Anc216_M5 is promising as a DON-detoxifying biocatalyst.
[前沿资讯 ] 魏茨曼科学研究所报道高效Kemp消除酶的完整计算设计 进入全文
科学网
魏茨曼科学研究所Sarel J. Fleishman研究团队报道了高效Kemp消除酶的完整计算设计。2025年6月18日，国际知名学术期刊《自然》发表了这一成果。 该研究团队提出了一个完全计算的工作流程，用于在TIM桶折叠中设计高效的酶，从天然蛋白质中提取主干片段，而无需通过突变文库筛选进行优化。三种Kemp消能设计的效率均大于 2,000 M−1 s−1。最有效的方法显示了来自任何天然蛋白质的140多个突变，包括一个新的活性位点。它具有高稳定性（大于85°C）和显著的催化效率（12,700 M−1 s−1）和速率（2.8s-1），比以前的计算设计高出两个数量级。 此外，设计一种在所有以前的Kemp消除设计中被认为是必不可少的残留物，可将效率提高到105 M−1 s−1，比率提高到30 s−1，实现与天然酶相当的催化参数，挑战基本的生物催化假设。通过克服设计方法上的限制，他们的策略使通过最小的实验努力就能编程出稳定、高效、全新的酶。 据了解，到目前为止，计算设计的酶表现出较低的催化速率，并且需要大量的实验优化才能达到与天然酶相似的活性水平。这些结果暴露了设计方法的局限性，并表明他们对生物催化基础知识的理解存在重大差距。
[学术文献 ] Chemoenzymatic platform with coordinated cofactor self-circulation for lignin valorization 进入全文
Nature Synthesis
Lignin valorization is vital for achieving economically viable and sustainable lignocellulosic biorefineries. However, the value-added utilization of lignin constituents is hampered by the requirement of expensive cofactors and low conversions. Here, by integrating coenzyme regeneration and cell-free expression, we report an in vitro multienzyme-coordinated expression with cofactor self-circulation (iMECS) strategy to achieve efficient lignin-to-molecule conversion. The iMECS system established a catalytic bridge with high atom economy for converting lignin waste into valuable aromatic compounds. Curcumin, vanillin and raspberry ketone were efficiently synthesized in a coenzyme-free manner, with a conversion of over 90%. We also demonstrated the flexibility of the iMECS platform, through which a wide array of phenylpropanoids could be easily obtained by enzyme swapping and pathway extension. By integrating chemical depolymerization with the iMECS system, lignin-rich agricultural waste was directly converted into valuable chemicals, and the overall catalytic efficiency was enhanced by up to 48-fold compared with the reported titres. This efficient, general platform can facilitate the utilization of lignocellulosic biomass, thereby promoting zero-waste biorefineries for a more sustainable future.
