Conley Erin, Wadler Caryn S, Bell Bailey A, Lucier Ivy, Haynie Caroline, Eldred Sophie, Nguyen Valerie, Bugni Tim S, Thomas Michael G
Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States.
Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States.
Biochemistry. 2024 Dec 3;63(23):3126-3135. doi: 10.1021/acs.biochem.4c00499. Epub 2024 Nov 21.
Nonribosomal peptide synthetases (NRPS) biosynthesize numerous natural products with therapeutic, agricultural, and industrial significance. Reliably altering substrate selection in these enzymes has been a longstanding goal, as this would enable the production of tailor-made peptides with desired activities. In this study, the NRPS EntF and the associated biosynthesis of the siderophore enterobactin (ENT) were used as a model system to interrogate substrate selection by an adenylation (A) domain. We employed a directed evolution pipeline that harnesses an genetic selection for siderophore production to alter A domain substrate selection. Surprisingly, this led to the formation of a new, physiologically active catechol siderophore in . We characterized the enzyme variants and demonstrated transferability of our findings to the well-studied TycC and GrsB NRPSs. This work identifies critical binding pocket residues that allow for altered substrate selection in our model system and expands upon our understanding of iron acquisition in .
非核糖体肽合成酶（NRPS）可生物合成众多具有治疗、农业和工业意义的天然产物。长期以来，可靠地改变这些酶中的底物选择一直是一个目标，因为这将能够生产出具有所需活性的定制肽。在本研究中，非核糖体肽合成酶EntF和铁载体肠杆菌素（ENT）的相关生物合成被用作一个模型系统，以探究腺苷化（A）结构域的底物选择。我们采用了一种定向进化流程，利用对铁载体产生的遗传筛选来改变A结构域的底物选择。令人惊讶的是，这导致在[具体环境或生物中]形成了一种新的、具有生理活性的儿茶酚铁载体。我们对酶变体进行了表征，并证明了我们的发现可转移到经过充分研究的TycC和GrsB非核糖体肽合成酶。这项工作确定了在我们的模型系统中允许改变底物选择的关键结合口袋残基，并扩展了我们对[具体生物或环境中]铁获取的理解。
