Patin Delphine, Bostock Julieanne, Blanot Didier, Mengin-Lecreulx Dominique, Chopra Ian
Université Paris-Sud, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, CNRS, Laboratoire des Enveloppes Bactériennes et Antibiotiques, UMR 8619, Orsay F-91405, France.
J Bacteriol. 2009 Dec;191(24):7430-5. doi: 10.1128/JB.01029-09. Epub 2009 Oct 9.
Chlamydiae are unusual obligately intracellular bacteria that do not synthesize detectable peptidoglycan. However, they possess genes that appear to encode products with peptidoglycan biosynthetic activity. Bioinformatic analysis predicts that chlamydial MurE possesses UDP-MurNAc-L-Ala-D-Glu:meso-diaminopimelic acid (UDP-MurNAc-L-Ala-D-Glu:meso-A(2)pm) ligase activity. Nevertheless, there are no experimental data to confirm this hypothesis. In this paper we demonstrate that the murE gene from Chlamydia trachomatis is capable of complementing a conditional Escherichia coli mutant impaired in UDP-MurNAc-L-Ala-D-Glu:meso-A(2)pm ligase activity. Recombinant MurE from C. trachomatis (MurE(Ct)) was overproduced in and purified from E. coli in order to investigate its kinetic parameters in vitro. By use of UDP-MurNAc-L-Ala-D-Glu as the nucleotide substrate, MurE(Ct) demonstrated ATP-dependent meso-A(2)pm ligase activity with pH and magnesium ion optima of 8.6 and 30 mM, respectively. Other amino acids (meso-lanthionine, the ll and dd isomers of A(2)pm, D-lysine) were also recognized by MurE(Ct.) However, the activities for these amino acid substrates were weaker than that for meso-A(2)pm. The specificity of MurE(Ct) for three possible C. trachomatis peptidoglycan nucleotide substrates was also determined in order to deduce which amino acid might be present at the first position of the UDP-MurNAc-pentapeptide. Relative k(cat)/K(m) ratios for UDP-MurNAc-L-Ala-D-Glu, UDP-MurNAc-L-Ser-D-Glu, and UDP-MurNAc-Gly-D-Glu were 100, 115, and 27, respectively. Our results are consistent with the synthesis in chlamydiae of a UDP-MurNAc-pentapeptide in which the third amino acid is meso-A(2)pm. However, due to the lack of specificity of MurE(Ct) for nucleotide substrates in vitro, it is not obvious which amino acid is present at the first position of the pentapeptide.
衣原体是一类特殊的专性胞内细菌，不合成可检测到的肽聚糖。然而，它们拥有一些似乎编码具有肽聚糖生物合成活性产物的基因。生物信息学分析预测衣原体的MurE具有UDP - N - 乙酰胞壁酸 - L - 丙氨酸 - D - 谷氨酸：内消旋二氨基庚二酸（UDP - MurNAc - L - Ala - D - Glu：meso - A(2)pm）连接酶活性。然而，尚无实验数据证实这一假设。在本文中，我们证明沙眼衣原体的murE基因能够互补一个在UDP - MurNAc - L - Ala - D - Glu：meso - A(2)pm连接酶活性方面受损的条件性大肠杆菌突变体。为了研究沙眼衣原体重组MurE（MurE(Ct)）的体外动力学参数，将其在大肠杆菌中过量表达并纯化。以UDP - MurNAc - L - Ala - D - Glu作为核苷酸底物时，MurE(Ct)表现出ATP依赖性的内消旋A(2)pm连接酶活性，最适pH值和镁离子浓度分别为8.6和30 mM。MurE(Ct)也能识别其他氨基酸（内消旋羊毛硫氨酸、A(2)pm的ll和dd异构体、D - 赖氨酸）。然而，这些氨基酸底物的活性比内消旋A(2)pm的活性弱。为了推断UDP - MurNAc - 五肽第一个位置可能存在哪种氨基酸，还测定了MurE(Ct)对三种可能的沙眼衣原体肽聚糖核苷酸底物的特异性。UDP - MurNAc - L - Ala - D - Glu、UDP - MurNAc - L - 丝氨酸 - D - 谷氨酸和UDP - MurNAc - 甘氨酸 - D - 谷氨酸的相对k(cat)/K(m)比值分别为100、115和27。我们的结果与衣原体中合成一种第三个氨基酸为内消旋A(2)pm的UDP - MurNAc - 五肽一致。然而，由于MurE(Ct)在体外对核苷酸底物缺乏特异性，五肽第一个位置存在哪种氨基酸并不明确。
