Maturation of IncP pilin precursors resembles the catalytic dyad-like mechanism of leader peptidases
Maturation of IncP pilin precursors resembles the catalytic dyad-like mechanism of leader peptidases
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DOI：
10.1128/jb.182.23.6751-6761.2000
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发表时间：
2000-12-01
影响因子：
3.2
通讯作者：
Lanka, E
中科院分区：
文献类型：
--
作者：
Eisenbrandt, R;Kalkum, M;Lanka, E
结合型IncP pill的菌毛亚基（菌毛蛋白）由trbC基因编码。IncP菌毛蛋白由78个氨基酸组成，形成环状结构（R. Eisenbrandt，M. Kalkum，E. M，Lai，C. I. Kado和E. Lanka，J.Biol.Chem.274：22548-22555，1999），三种酶参与菌毛蛋白的成熟：用于去除信号肽的大肠杆菌的LepB和用于去除27个C末端残基的尚未鉴定的蛋白酶。这两种酶都是染色体编码的。最后，内膜结合的IncP TraF用截短的N末端替换四个氨基酸的C末端肽，产生环状多肽，我们将后一个过程称为“前菌毛蛋白环化”。我们已经使用trbC和traF的定点突变来解开菌毛蛋白成熟过程。采用基质辅助激光解吸电离质谱法，以反式-3-吲哚丙烯酸（trans-3-indolyl acrylate，TA）为基质，采用优化的全细胞样品制备技术，测定了Prepilin的有效环化。我们发现，在TrbC核心序列中的几个氨基酸交换允许prepilin环化，但禁用随后的菌毛组装。我们提出了一种机制，解释如何信号肽酶同源TraF攻击的C-末端部分的TrbC核心序列通过激活的丝氨酸残基。而不是切割和释放水解肽，TraF大概反应作为肽基转移酶，涉及N末端的TrbC在氨解的一个假定的TraF-乙酰基-TrbC中间体。在C-末端四肽的形式损失下，在协同作用中形成新的肽键，将TrbC的丝氨酸37与甘氨酸114连接。
The pilus subunit, the pilin, of conjugative IncP pill is encoded by the trbC gene. IncP pilin is composed of 78 amino acids forming a ring structure (R. Eisenbrandt, M. Kalkum, E.-M, Lai, C. I. Kado, and E. Lanka, J. Biol. Chem. 274:22548-22555, 1999), Three enzymes are involved in maturation of the pilin: LepB of Escherichia coli for signal peptide removal and a yet-unidentified protease for removal of 27 C terminal residues. Both enzymes are chromosome encoded. Finally, the inner membrane-associated IncP TraF replaces a four-amino-acid C-terminal peptide with the truncated N terminus, yielding the cyclic polypeptide, We refer to the latter process as "prepilin cyclization," We have used site-directed mutagenesis of trbC and traF to unravel the pilin maturation process, Each of the mutants was analyzed for its phenotypes of prepilin cyclization, pilus formation, donor-specific phage adsorption, and conjugative DNA transfer abilities, Effective prepilin cyclization was determined by matrix-assisted laser desorption-ionization-mass spectrometry using an optimized sample preparation technique of whole cells and trans-3-indolyl acrylic acid as a matrix. We found that several amino acid exchanges in the TrbC core sequence allow prepilin cyclization but disable the succeeding pilus assembly. We propose a mechanism explaining how the signal peptidase homologue TraF attacks a C-terminal section of the TrbC core sequence via an activated serine residue. Rather than cleaving and releasing hydrolyzed peptides, TraF presumably reacts as a peptidyl transferase, involving the N terminus of TrbC in the aminolysis of a postulated TraF-acetyl-TrbC intermediate. Under formal loss of a C-terminal tetrapeptide, a new peptide bond is formed in a concerted action, connecting serine 37 with glycine 114 of TrbC.