Paetzel M, Dalbey R E, Strynadka N C
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
Nature. 1998 Nov 12;396(6707):186-90. doi: 10.1038/24196.
The signal peptidase (SPase) from Escherichia coli is a membrane-bound endopeptidase with two amino-terminal transmembrane segments and a carboxy-terminal catalytic region which resides in the periplasmic space. SPase functions to release proteins that have been translocated into the inner membrane from the cell interior, by cleaving off their signal peptides. We report here the X-ray crystal structure of a catalytically active soluble fragment of E. coli SPase (SPase delta2-75). We have determined this structure at 1.9 A resolution in a complex with an inhibitor, a beta-lactam (5S,6S penem), which is covalently bound as an acyl-enzyme intermediate to the gamma-oxygen of a serine residue at position 90, demonstrating that this residue acts as the nucleophile in the hydrolytic mechanism of signal-peptide cleavage. The structure is consistent with the use by SPase of Lys 145 as a general base in the activation of the nucleophilic Ser90, explains the specificity requirement at the signal-peptide cleavage site, and reveals a large exposed hydrophobic surface which could be a site for an intimate association with the membrane. As enzymes that are essential for cell viability, bacterial SPases present a feasible antibacterial target: our determination of the SPase structure therefore provides a template for the rational design of antibiotic compounds.
来自大肠杆菌的信号肽酶（SPase）是一种膜结合的内肽酶，具有两个氨基末端跨膜片段和一个位于周质空间的羧基末端催化区域。SPase的功能是通过切割已从细胞内部转运到内膜中的蛋白质的信号肽，从而释放这些蛋白质。我们在此报告大肠杆菌SPase（SPase delta2-75）的催化活性可溶性片段的X射线晶体结构。我们已在1.9埃分辨率下确定了该结构，它与一种抑制剂——β-内酰胺（5S，6S青霉烯）形成复合物，该β-内酰胺作为酰基酶中间体与90位丝氨酸残基的γ-氧共价结合，这表明该残基在信号肽切割的水解机制中作为亲核试剂起作用。该结构与SPase使用赖氨酸145作为亲核丝氨酸90激活的通用碱一致，解释了信号肽切割位点的特异性要求，并揭示了一个大的暴露疏水表面，该表面可能是与膜紧密结合的位点。作为对细胞活力至关重要的酶，细菌SPase是一个可行的抗菌靶点：因此我们对SPase结构的测定为合理设计抗生素化合物提供了一个模板。
