Strom M S, Nunn D N, Lory S
Utilization Research Division, Northwest Fisheries Science Center, NMFS, NOAA, Seattle, Washington 98112.
Methods Enzymol. 1994;235:527-40. doi: 10.1016/0076-6879(94)35168-6.
We have described the characterization of a protein initially identified as having an essential function in biogenesis of polar pili of P. aeruginosa by processing precursors of pilin. Other findings have also expanded the range of substrates for PilD to include a set of proteins that are essential components of the extracellular secretion machinery. Direct demonstration of prepilin processing necessitates use of purified substrates and enzymes, and we present general protocols for purification of both enzymes and substrates, as well as an assay for prepilin peptidase activity. For a source of enzyme and substrates, mutants of P. aeruginosa defective in pilin processing as well as clones overexpressing the pilin gene and PilD were developed. These methods are applicable to other bacterial systems that express Type IV pili and/or possess the PilD-dependent machinery of extracellular protein secretion. PilD is a bifunctional enzyme, which carries out not only cleavage but also amino-terminal methylation of the mature pilin. Cleavage and N-methylation of the pilin-like Xcp proteins involved in extracellular protein secretion have also been shown to be dependent on PilD. The leader peptidase activity of PilD is inhibited by sulfhydryl blocking reagents such as NEM and PCMB, whereas the methyltransferase activity of the purified enzyme is dependent on reduction with dithiothreitol. The conserved region containing the cysteine residues lies within the largest hydrophilic domain of the protein as predicted from hydrophobicity analysis, and it is probably exposed to the cytoplasmic side of the cytoplasmic membrane. Identification of the active site residues involved in recognition of the substrates for processing and subsequent methylation is currently underway. Studies on substrate specificities of PilD, with respect to its leader peptidase and methyltransferase activity, may prove to be useful in designing inhibitors which would interfere with maturation of Type IV prepilins and components of the extracellular protein secretion machinery. In light of the fact that an increasing number of both mammalian and plant pathogens are being shown to have extracellular secretion pathways homologous to that seen for P. aeruginosa, such inhibitors may be useful tools in the study of the role these peptidases play in bacterial virulence.
我们已经描述了一种蛋白质的特性，该蛋白质最初被鉴定为通过加工菌毛蛋白前体在铜绿假单胞菌极毛生物合成中具有重要功能。其他研究结果还扩大了PilD的底物范围，使其包括细胞外分泌机制的一组必需蛋白质。菌毛蛋白前体加工的直接证明需要使用纯化的底物和酶，我们提供了纯化酶和底物的通用方案，以及菌毛蛋白肽酶活性的测定方法。为了获得酶和底物的来源，我们构建了菌毛蛋白加工缺陷的铜绿假单胞菌突变体以及过表达菌毛蛋白基因和PilD的克隆。这些方法适用于其他表达IV型菌毛和/或拥有依赖PilD的细胞外蛋白质分泌机制的细菌系统。PilD是一种双功能酶，它不仅进行切割，还对成熟菌毛蛋白进行氨基末端甲基化。参与细胞外蛋白质分泌的类菌毛蛋白Xcp蛋白的切割和N-甲基化也已被证明依赖于PilD。PilD的前导肽酶活性受到巯基阻断试剂如NEM和PCMB的抑制，而纯化酶的甲基转移酶活性则依赖于二硫苏糖醇的还原作用。根据疏水性分析预测，含有半胱氨酸残基的保守区域位于该蛋白质最大的亲水区内，并且可能暴露于细胞质膜的细胞质一侧。目前正在确定参与识别加工底物和后续甲基化的活性位点残基。关于PilD的前导肽酶和甲基转移酶活性的底物特异性研究，可能有助于设计干扰IV型菌毛蛋白前体和细胞外蛋白质分泌机制成分成熟的抑制剂。鉴于越来越多的哺乳动物和植物病原体被证明具有与铜绿假单胞菌相似的细胞外分泌途径，此类抑制剂可能是研究这些肽酶在细菌毒力中作用的有用工具。
