Schemenauer Daniel E, Pool Emily H, Raynor Stephanie N, Ruiz Gabriela P, Goehring Leah M, Koelper Andrew J, Wilson Madeleine A, Durand Anthony J, Kourtoglou Elexi C, Larsen Erik M, Lavis Luke D, Esteb John J, Hoops Geoffrey C, Johnson R Jeremy
Department of Chemistry and Biochemistry, Butler University, Indianapolis, Indiana 46208, United States.
Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, Virginia 20147, United States.
ACS Omega. 2023 Mar 30;8(14):13252-13264. doi: 10.1021/acsomega.3c00534. eCollection 2023 Apr 11.
has a complex life cycle transitioning between active and dormant growth states depending on environmental conditions. LipN (Rv2970c) is a conserved mycobacterial serine hydrolase with regulated catalytic activity at the interface between active and dormant growth conditions. LipN also catalyzes the xenobiotic degradation of a tertiary ester substrate and contains multiple conserved motifs connected with the ability to catalyze the hydrolysis of difficult tertiary ester substrates. Herein, we expanded a library of fluorogenic ester substrates to include more tertiary and constrained esters and screened 33 fluorogenic substrates for activation by LipN, identifying its unique substrate signature. LipN preferred short, unbranched ester substrates, but had its second highest activity against a heteroaromatic five-membered oxazole ester. Oxazole esters are present in multiple mycobacterial serine hydrolase inhibitors but have not been tested widely as ester substrates. Combined structural modeling, kinetic measurements, and substitutional analysis of LipN showcased a fairly rigid binding pocket preorganized for catalysis of short ester substrates. Substitution of diverse amino acids across the binding pocket significantly impacted the folded stability and catalytic activity of LipN with two conserved motifs (HGGGW and GDSAG) playing interconnected, multidimensional roles in regulating its substrate specificity. Together this detailed substrate specificity profile of LipN illustrates the complex interplay between structure and function in mycobacterial hormone-sensitive lipase homologues and indicates oxazole esters as promising inhibitor and substrate scaffolds for mycobacterial hydrolases.
根据环境条件，其具有在活跃生长状态和休眠生长状态之间转换的复杂生命周期。LipN（Rv2970c）是一种保守的分枝杆菌丝氨酸水解酶，在活跃生长条件和休眠生长条件之间的界面处具有受调控的催化活性。LipN还催化叔酯底物的异源生物降解，并包含多个与催化难水解叔酯底物水解能力相关的保守基序。在此，我们扩展了荧光酯底物库，以纳入更多叔酯和受限酯，并筛选了33种荧光底物以检测其是否被LipN激活，从而确定其独特的底物特征。LipN偏好短的、无支链的酯底物，但对一种杂芳族五元恶唑酯的活性次之。恶唑酯存在于多种分枝杆菌丝氨酸水解酶抑制剂中，但尚未作为酯底物进行广泛测试。结合结构建模、动力学测量和LipN的取代分析表明，存在一个相当刚性的结合口袋，预先组织好用于催化短酯底物。在整个结合口袋中替换不同氨基酸会显著影响LipN的折叠稳定性和催化活性，两个保守基序（HGGGW和GDSAG）在调节其底物特异性方面发挥着相互关联的多维作用。LipN的这一详细底物特异性概况共同说明了分枝杆菌激素敏感脂肪酶同源物中结构与功能之间的复杂相互作用，并表明恶唑酯是分枝杆菌水解酶有前景的抑制剂和底物支架。
