The MurE synthetase from Thermotoga maritima is endowed with an unusual D-lysine adding activity
The MurE synthetase from Thermotoga maritima is endowed with an unusual D-lysine adding activity
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DOI：
10.1074/jbc.m506311200
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发表时间：
2006-06-09
影响因子：
4.8
通讯作者：
Blanot, Didier
中科院分区：
文献类型：
--
作者：
Boniface, Audrey;Bouhss, Ahmed;Blanot, Didier
海栖热袍菌（Thermotoga maritima）（一种极端嗜热真细菌）的肽聚糖显示不含二氨基庚二酸和近似量的赖氨酸的两种对映异构体（Huber，R.，Langworthy，T.一、Konig，H.，M.，韦斯角，澳-地R.，斯莱特尔大学B.，和Stetter，K. O.（1986）Arch.Microbiol. 144、324和333）。为了评估可能参与的MurE活性的D-赖氨酸的掺入，从这个生物体的murE基因克隆在大肠杆菌中，并纯化相应的蛋白质作为C-末端His 6-标记的形式。体外试验表明，D-赖氨酸和内消旋-二氨基庚二酸被添加到UDP-N-乙酰胞壁酰二肽中，相对于L-赖氨酸，效率分别为25%和10%。纯化的酶用于合成含L-和D-赖氨酸的UDP-N-乙酰胞壁酰三肽;化学分析揭示了含D-赖氨酸的核苷酸的不寻常结构，即D-赖氨酸的ε-氨基功能被D-谷氨酰残基酰化。用T. maritima的研究表明，这种新的核苷酸不是MurF的底物，但它可以通过MraY直接加工成三肽脂质I，从而证实了MurE在D-赖氨酸掺入肽聚糖中的作用。
The peptidoglycan of Thermotoga maritima, an extremely thermophilic eubacterium, was shown to contain no diaminopimelic acid and approximate amounts of both enantiomers of lysine (Huber, R., Langworthy, T. A., Konig, H., Thomm, M., Woese, C. R., Sleytr, U. B., and Stetter, K.O. (1986) Arch. Microbiol. 144, 324 333). To assess the possible involvement of the MurE activity in the incorporation of D-lysine, the murE gene from this organism was cloned in Escherichia coli, and the corresponding protein was purified as the C-terminal His6-tagged form. In vitro assays showed that D-lysine and meso-diaminopimelic acid were added to UDP-N-acetylmuramoyldipeptide with 25 and 10% efficiencies, respectively, relative to L-lysine. The purified enzyme was used to synthesize the L- and D-lysine-containing UDP-N-acetylmuramoyl-tripeptides; chemical analysis revealed an unusual structure for the D-lysine-containing nucleotide, namely acylation of the epsilon-amino function of D-lysine by the D-glutamyl residue. In vitro assays with MurF and MraY enzymes from T. maritima showed that this novel nucleotide was not a substrate for MurF but that it could be directly processed into tripeptide lipid I by MraY, thereby substantiating the role of MurE in the incorporation of D-lysine into peptidoglycan.