BACTERIAL ANCESTORS OF ENZYMES INVOLVED IN UBIQUITIN-LIKE PROTEIN CONJUGATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 微球菌素是一种修饰的多肽，可以抑制竞争对手的革兰氏阴性细菌的生长，如大肠杆菌、沙门氏菌和肠杆菌。微菌素C7(MccC7)是由大肠杆菌产生的，通过一种“特洛伊木马”机制消灭竞争对手。在导入靶细胞后，MccC7被非特异性多肽酶切割，释放出一种针对天冬氨酰-tRNA合成酶的有毒的腺苷化天冬氨酸模拟物。MccC7是通过修饰前体七肽MCCA(序列MRTGNAN)产生的。MccC7生物合成的翻译后步骤包括将C端的Asn7转化为天冬氨酸酰胺，并将氮磷酰胺连接到AMP上，然后是磷酸氧氨丙基化。Asn7氨基氮的迁移和N-P键的形成步骤是由MccB酶催化的。在消耗一个三磷酸腺苷的传统的酰基腺基化反应中，MccB催化MCCA C末端的腺基化。分子内重排后，形成MCCA多肽-丁二酰亚胺中间体。接下来，MccB催化琥珀酰亚胺的一种不寻常的腺基化：琥珀酰亚胺氮攻击第二个ATP分子的磷酸，通过N-P键将AMP连接到多肽末端。琥珀酰亚胺环通过区域特异性的水介导的开孔被水解，生成多肽-酰基-N-P-腺苷基团，即特洛伊木马试剂。 MccB的N末端~90残基区域与已知结构没有可检测到的同源性。MccB的C末端~260个残基与部分泛素样蛋白(UBL)激活酶有同源性，也称为E1S，启动UBL结合。这种序列相似性提出了关于MccB和E1机制的共同和不同方面的几个问题。首先，MccB如何识别七肽底物，而其他家族成员有E8 kDa Ubl底物？第二，为什么MccB催化多肽C-末端ASN或琥珀酰亚胺的腺基化，而不是UBLC-末端的Gly-Gly序列？第三，MccB如何催化两个连续的腺基化反应？为了解决这些问题，我们分析了MccB-MCCA-核苷酸相互作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Microcins are modified peptides that inhibit growth of competing Gram-negative bacteria such as Escherichia, Salmonella, and Enterobacter. Microcin C7 (MccC7) is produced by E. coli to eradicate competitors through a "Trojan horse" mechanism. After import into target cells, MccC7 is cleaved by nonspecific peptidases to release a toxic adenylated-aspartic-acid mimic that targets aspartyl-tRNA synthetase. MccC7 is generated by modification of a precursor heptapeptide, MccA (sequence MRTGNAN). Posttranslational steps in MccC7 biosynthesis involve conversion of the C-terminal Asn7 to an Asp amide with the nitrogen phosphoramidate-linked to AMP, followed by aminopropylation of a phosphate oxygen. Migration of the Asn7 carboxamido nitrogen and the N-P bond-forming step are catalyzed by the enzyme MccB. In a conventional acyl-adenylation reaction that consumes one ATP, MccB catalyzes adenylation of the MccA C-terminus. After an intramolecular rearrangement, an MccA peptidyl-succinimide intermediate is formed. Next, MccB catalyzes an unusual adenylation of the succinimide: the succinimidyl nitrogen attacks the ¿-phosphate of a second ATP molecule, linking AMP to the peptide terminus via an N-P bond. The succinimide ring is hydrolyzed by regiospecific water-mediated opening to yield to the peptidyl-acyl-N-P-Adenosine group that is the Trojan horse reagent. MccB's N-terminal ~90 residue region is not detectably homologous to known structures. MccB's C-terminal ~260 residues share homology with a portion of ubiquitin-like protein (UBL) activating enzymes, also called E1s, which initiate UBL conjugation. This sequence similarity raises several questions about common and divergent aspects of MccB and E1 mechanisms. First, how does MccB recognize a heptapeptide substrate, whereas other family members have a e8 kDa UBL substrate? Second, why does MccB catalyze adenylation of a peptide C-terminal Asn or succinimide, rather than the Gly-Gly sequence found at UBL C-termini? Third, how can MccB catalyze two successive adenylation reactions? To address these questions, we analyzed MccB-MccA-nucleotide interactions.