Drufva Erin E, Cahill John F, Saint-Vincent Patricia M B, Williams Alexis N, Bocharova Vera, Capra Nikolas, Meilleur Flora, Carper Dana L, Bourgery Célestin, Miyazaki Kaito, Yonemura Maina, Shiraishi Yuki, Parks Jerry M, Zhou Muchu, Dishner Isaiah T, Foster Jeffrey C, Koehler Stephen J, Valentino Hannah R, Sedova Ada, Kertesz Vilmos, Vasileva Delyana P, Hochanadel Leah H, Figg C Adrian, Negoro Seiji, Kato Dai-Ichiro, Chen Serena H, Michener Joshua K
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA 37830.
Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, Oak Ridge, TN, USA 37830.
bioRxiv. 2024 Nov 14:2024.11.14.623603. doi: 10.1101/2024.11.14.623603.
Enzymes have evolved to rapidly and selectively hydrolyze diverse natural and anthropogenic polymers, but only a limited group of related enzymes have been shown to hydrolyze synthetic polyamides. In this work, we synthesized and characterized a panel of 95 diverse enzymes from the N-terminal nucleophile hydrolase superfamily with 30-50% pairwise amino acid identity. We found that nearly 40% of the enzymes had substantial nylon hydrolase activity, in many cases comparable to that of the best-characterized nylon hydrolase, NylC. There was no relationship between phylogeny and activity, nor any evidence of prior selection for nylon hydrolase activity. Several newly-identified hydrolases showed significant substrate selectivity, generating up to 20-fold higher product titers with Nylon 6,6 versus Nylon 6. Finally, we determined the crystal structure and oligomerization state of a Nylon 6,6-selective hydrolase to elucidate structural factors that could affect activity and selectivity. These new enzymes provide insights into the widespread potential for nylon hydrolase evolution and opportunities for analysis and engineering of improved hydrolases.
酶已经进化到能够快速且有选择性地水解各种天然和人为合成的聚合物，但只有有限的一组相关酶被证明能够水解合成聚酰胺。在这项工作中，我们合成并表征了一组来自N端亲核水解酶超家族的95种不同的酶，它们的氨基酸序列两两之间有30%-50%的同源性。我们发现，近40%的酶具有显著的尼龙水解酶活性，在许多情况下，其活性与表征最充分的尼龙水解酶NylC相当。系统发育与活性之间没有关联，也没有任何先前针对尼龙水解酶活性进行选择的证据。几种新鉴定的水解酶表现出显著的底物选择性，相对于尼龙6，它们对尼龙6,6产生的产物滴度高达20倍。最后，我们确定了一种尼龙6,6选择性水解酶的晶体结构和寡聚状态，以阐明可能影响活性和选择性的结构因素。这些新酶为尼龙水解酶进化的广泛潜力以及改进水解酶的分析和工程设计提供了见解。
