Chlamydia trachomatis dapF Encodes a Bifunctional Enzyme Capable of Both d-Glutamate Racemase and Diaminopimelate Epimerase Activities.
Chlamydia trachomatis dapF Encodes a Bifunctional Enzyme Capable of Both d-Glutamate Racemase and Diaminopimelate Epimerase Activities.
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DOI：
10.1128/mbio.00204-18
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发表时间：
2018-04-03
期刊：
影响因子：
6.4
通讯作者：
Maurelli AT
中科院分区：
文献类型：
--
作者：
Liechti G;Singh R;Rossi PL;Gray MD;Adams NE;Maurelli AT
肽聚糖是细菌特有的糖/氨基酸聚合物，对于分裂和细胞形状维持至关重要。构成其交联茎肽的 d-氨基酸在自然界中并不丰富，必须由细菌从头合成。 d-谷氨酸存在于五肽茎的第二个位置，并且在所有细菌物种中严格保守。在革兰氏阴性细菌中，D-谷氨酸是通过谷氨酸消旋酶 (MurI) 使 L-谷氨酸外消旋而产生的。沙眼衣原体是全世界传染性失明和性传播细菌感染的主要原因。虽然其基因组编码参与肽聚糖合成的大部分酶，但尚未注释过 murI 同源物。最近的研究揭示了沙眼衣原体中存在肽聚糖，并证实其五肽包含 d-谷氨酸。在这项研究中，我们证明沙眼衣原体利用二氨基庚二酸差向异构酶 (DapF) 的新型双功能同源物合成 d-谷氨酸。 DapF 催化内消旋二氨基庚二酸（肽聚糖特有的另一种氨基酸）合成的最后一步。大肠杆菌 murI 突变体的基因互补证明衣原体 DapF 可以产生 d-谷氨酸。生化分析显示出强大的活性，但与典型的谷氨酸消旋酶不同，其活性依赖于辅因子磷酸吡哆醛。遗传互补、酶学表征和生物信息学分析表明，衣原体 DapF 与其他混杂/原始酶具有相同的特征，这不仅在衣原体中，而且在浮霉菌-疣微菌-衣原体总门中缺乏公认的谷氨酸消旋酶的许多其他属中提供了 d-谷氨酸合成的潜在机制。在这里，我们描述了致病性衣原体物种肽聚糖合成中最后剩下的“缺失”步骤之一，即 d-谷氨酸的合成。我们已经确定，沙眼衣原体编码的二氨基庚二酸差向异构酶（DapF）能够进行DAP的差向异构化和谷氨酸的磷酸吡哆醛依赖性外消旋化。酶的混杂被认为是这个星球上早期微生物生命的标志，目前关于“兼职酶”是否代表原始进化遗迹，还是最近还原论进化压力的产物，目前存在着激烈的争论。鉴于大量衣原体物种（以及浮霉菌-疣状微生物-衣原体超门的成员）拥有 DapF 但缺乏 MurI 同源物，DapF 很可能是一种原始异构酶，在这些生物体中既充当消旋酶又充当差向异构酶，这表明专门的 d-谷氨酸消旋酶从未在这些微生物中进化。
Peptidoglycan is a sugar/amino acid polymer unique to bacteria and essential for division and cell shape maintenance. The d-amino acids that make up its cross-linked stem peptides are not abundant in nature and must be synthesized by bacteria de novo. d-Glutamate is present at the second position of the pentapeptide stem and is strictly conserved in all bacterial species. In Gram-negative bacteria, d-glutamate is generated via the racemization of l-glutamate by glutamate racemase (MurI). Chlamydia trachomatis is the leading cause of infectious blindness and sexually transmitted bacterial infections worldwide. While its genome encodes a majority of the enzymes involved in peptidoglycan synthesis, no murI homologue has ever been annotated. Recent studies have revealed the presence of peptidoglycan in C. trachomatis and confirmed that its pentapeptide includes d-glutamate. In this study, we show that C. trachomatis synthesizes d-glutamate by utilizing a novel, bifunctional homologue of diaminopimelate epimerase (DapF). DapF catalyzes the final step in the synthesis of meso-diaminopimelate, another amino acid unique to peptidoglycan. Genetic complementation of an Escherichia coli murI mutant demonstrated that Chlamydia DapF can generate d-glutamate. Biochemical analysis showed robust activity, but unlike canonical glutamate racemases, activity was dependent on the cofactor pyridoxal phosphate. Genetic complementation, enzymatic characterization, and bioinformatic analyses indicate that chlamydial DapF shares characteristics with other promiscuous/primordial enzymes, presenting a potential mechanism for d-glutamate synthesis not only in Chlamydia but also numerous other genera within the Planctomycetes-Verrucomicrobiae-Chlamydiae superphylum that lack recognized glutamate racemases. Here we describe one of the last remaining “missing” steps in peptidoglycan synthesis in pathogenic Chlamydia species, the synthesis of d-glutamate. We have determined that the diaminopimelate epimerase (DapF) encoded by Chlamydia trachomatis is capable of carrying out both the epimerization of DAP and the pyridoxal phosphate-dependent racemization of glutamate. Enzyme promiscuity is thought to be the hallmark of early microbial life on this planet, and there is currently an active debate as to whether “moonlighting enzymes” represent primordial evolutionary relics or are a product of more recent reductionist evolutionary pressures. Given the large number of Chlamydia species (as well as members of the Planctomycetes-Verrucomicrobiae-Chlamydiae superphylum) that possess DapF but lack homologues of MurI, it is likely that DapF is a primordial isomerase that functions as both racemase and epimerase in these organisms, suggesting that specialized d-glutamate racemase enzymes never evolved in these microbes.