Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218.
Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218
Proc Natl Acad Sci U S A. 2021 Apr 27;118(17). doi: 10.1073/pnas.2026017118.
Nonribosomal peptide synthetases (NRPSs) are large, multidomain biosynthetic enzymes involved in the assembly-line-like synthesis of numerous peptide natural products. Among these are clinically useful antibiotics including three classes of β-lactams: the penicillins/cephalosporins, the monobactams, and the monocyclic nocardicins, as well as the vancomycin family of glycopeptides and the depsipeptide daptomycin. During NRPS synthesis, peptide bond formation is catalyzed by condensation (C) domains, which couple the nascent peptide with the next programmed amino acid of the sequence. A growing number of additional functions are linked to the activity of C domains. In the biosynthesis of the nocardicins, a specialized C domain prepares the embedded β-lactam ring from a serine residue. Here, we examine the evolutionary descent of this unique β-lactam-synthesizing C domain. Guided by its ancestry, we predict and demonstrate in vitro that this C domain alternatively performs peptide bond formation when a single stereochemical change is introduced into its peptide starting material. Remarkably, the function of the downstream thioesterase (TE) domain also changes. Natively, the TE directs C terminus epimerization prior to hydrolysis when the β-lactam is made but catalyzes immediate release of the alternative peptide. In addition, we investigate the roles of C-domain histidine residues in light of clade-specific sequence motifs, refining earlier mechanistic proposals of both β-lactam formation and canonical peptide synthesis. Finally, expanded phylogenetic analysis reveals unifying connections between β-lactam synthesis and allied C domains associated with the appearance of ᴅ-amino acid and dehydroamino acid residues in other NRPS-derived natural products.
非核糖体肽合成酶（NRPSs）是一类大型的、多结构域的生物合成酶，参与众多肽类天然产物的装配线式合成。其中包括临床上有用的抗生素，包括三类β-内酰胺类抗生素：青霉素/头孢菌素、单环β-内酰胺类抗生素和单环诺卡菌素，以及万古霉素类糖肽和去甲万古霉素类脂肽抗生素。在 NRPS 合成过程中，肽键的形成由缩合（C）结构域催化，该结构域将新生肽与序列中下一个编程的氨基酸偶联。越来越多的其他功能与 C 结构域的活性相关联。在诺卡菌素的生物合成中，一个特殊的 C 结构域从丝氨酸残基中制备嵌入的β-内酰胺环。在这里，我们研究了这个独特的β-内酰胺合成 C 结构域的进化来源。根据其祖先，我们预测并在体外证明，当其肽起始材料发生单一立体化学变化时，该 C 结构域可以替代地进行肽键形成。值得注意的是，下游硫酯酶（TE）结构域的功能也发生了变化。天然情况下，当β-内酰胺合成时，TE 会在水解前引导 C 末端差向异构化，但会立即释放替代肽。此外，我们还根据特定进化枝的序列基序研究了 C 结构域组氨酸残基的作用，从而改进了之前关于β-内酰胺形成和典型肽合成的机制假设。最后，扩展的系统发育分析揭示了β-内酰胺合成与其他 NRPS 衍生天然产物中出现的ᴅ-氨基酸和脱氢氨基酸残基相关联的 C 结构域之间的统一联系。
