Jackson Sophie N, Lee Darren E, Blount Jadon M, Croney Kayla A, Ibershof Justin W, Ceravolo Caroline M, Brown Kate M, Goodwin-Rice Noah J, Whitham Kyle M, McCarty James, Antos John M, Amacher Jeanine F
Department of Chemistry, Western Washington University, Bellingham, Washington, USA.
Department of Chemistry, Western Washington University, Bellingham, Washington, USA.
J Biol Chem. 2025 Apr;301(4):108382. doi: 10.1016/j.jbc.2025.108382. Epub 2025 Mar 4.
Sortases are critical cysteine transpeptidases that facilitate the attachment of proteins to the cell wall in Gram-positive bacteria. These enzymes are potential targets for novel antibiotic development, and versatile tools in protein engineering applications. There are six classes of sortases recognized, yet class A sortases (SrtA) are the most widely studied and utilized. SrtA enzymes endogenously recognize the amino acid sequence LPXTG, where X = any amino acid, with additional promiscuity now recognized in multiple positions for certain SrtA enzymes. Much less is known about Class B sortases (SrtB), which target a distinct sequence, typically with an N-terminal Asn, e.g., variations of NPXTG or NPQTN. Although understudied overall, two SrtB enzymes were previously shown to be specific for heme transporter proteins, and in vitro experiments with the catalytic domains of these enzymes reveal activities significantly worse than SrtA from the same organisms. Here, we use protein biochemistry, structural analyses, and computational simulations to better understand and characterize these enzymes, specifically investigating Bacillus anthracis SrtB (baSrtB) as a model SrtB protein. Structural modeling predicts a plausible enzyme-substrate complex, which is verified by mutagenesis of binding cleft residues. Furthermore, residues N- and C-terminal to the pentapeptide recognition motif are critical for observed activity. Finally, we use chimeric proteins to identify mutations that improve baSrtB activity by ∼4-fold, and demonstrate the feasibility of sortase-mediated ligation using a baSrtB enzyme variant. These studies provide insight into SrtB-target binding as well as evidence that SrtB enzymes can be modified to be of potential use in protein engineering.
分选酶是关键的半胱氨酸转肽酶，可促进革兰氏阳性菌中蛋白质与细胞壁的附着。这些酶是新型抗生素开发的潜在靶点，也是蛋白质工程应用中的多功能工具。已识别出六类分选酶，但A类分选酶（SrtA）是研究和应用最为广泛的。SrtA酶可内源性识别氨基酸序列LPXTG，其中X = 任何氨基酸，现在还发现某些SrtA酶在多个位置具有额外的识别选择性。对于B类分选酶（SrtB）的了解则少得多，其靶向不同的序列，通常具有N端天冬酰胺，例如NPXTG或NPQTN的变体。尽管总体上研究较少，但先前已证明两种SrtB酶对血红素转运蛋白具有特异性，对这些酶催化结构域的体外实验表明，其活性明显低于同一生物体中的SrtA。在此，我们运用蛋白质生物化学、结构分析和计算模拟来更好地理解和表征这些酶，具体以炭疽芽孢杆菌SrtB（baSrtB）作为模型SrtB蛋白进行研究。结构建模预测了一种合理的酶-底物复合物，通过对结合裂隙残基进行诱变得到了验证。此外，五肽识别基序N端和C端的残基对观察到的活性至关重要。最后，我们使用嵌合蛋白来鉴定可将baSrtB活性提高约4倍的突变，并证明使用baSrtB酶变体进行分选酶介导连接的可行性。这些研究为SrtB-靶点结合提供了见解，也证明了SrtB酶可经修饰后在蛋白质工程中具有潜在用途。
