CNRS, Aix-Marseille Université, Enzymologie Interfaciale et Physiologie de la Lipolyse UPR 9025, 31 chemin Joseph Aiguier, 13402 Marseille cedex 20, France.
J Bacteriol. 2010 Sep;192(18):4776-85. doi: 10.1128/JB.00261-10. Epub 2010 Jul 2.
MSMEG_0220 from Mycobacterium smegmatis, the ortholog of the Rv0183 gene from M. tuberculosis, recently identified and characterized as encoding a monoacylglycerol lipase, was cloned and expressed in Escherichia coli. The recombinant protein (rMSMEG_0220), which exhibits 68% amino acid sequence identity with Rv0183, showed the same substrate specificity and similar patterns of pH-dependent activity and stability as the M. tuberculosis enzyme. rMSMEG_0220 was found to hydrolyze long-chain monoacylglycerol with a specific activity of 143 +/- 6 U mg(-1). Like Rv0183 in M. tuberculosis, MSMEG_0220 was found to be located in the cell wall. To assess the in vivo role of the homologous proteins, an MSMEG_0220 disrupted mutant of M. smegmatis (MsDelta0220) was produced. An intriguing change in the colony morphology and in the cell interaction, which were partly restored in the complemented mutant containing either an active (ComMsDelta0220) or an inactive (ComMsDelta0220S111A) enzyme, was observed. Growth studies performed in media supplemented with monoolein showed that the ability of both MsDelta0220 and ComMsDelta0220S111A to grow in the presence of this lipid was impaired. Moreover, studies of the antimicrobial susceptibility of the MsDelta0220 strain showed that this mutant is more sensitive to rifampin and more resistant to isoniazid than the wild-type strain, pointing to a critical structural role of this enzyme in mycobacterial physiology, in addition to its function in the hydrolysis of exogenous lipids.
从耻垢分枝杆菌中鉴定并表征的 MSMEG_0220 是 M. tuberculosis 的 Rv0183 基因的同源物，它编码一种单酰基甘油脂肪酶。该基因在大肠杆菌中克隆和表达。重组蛋白（rMSMEG_0220）与 Rv0183 具有 68%的氨基酸序列同一性，表现出相同的底物特异性和相似的 pH 依赖性活性和稳定性模式，与结核分枝杆菌酶相似。rMSMEG_0220 被发现能够水解长链单酰基甘油，比活为 143 ± 6 U mg(-1)。与结核分枝杆菌中的 Rv0183 一样，MSMEG_0220 被发现位于细胞壁中。为了评估同源蛋白的体内作用，构建了耻垢分枝杆菌 MSMEG_0220 缺失突变株（MsDelta0220）。令人感兴趣的是，观察到菌落形态和细胞相互作用发生了变化，这些变化在含有活性（ComMsDelta0220）或失活（ComMsDelta0220S111A）酶的互补突变体中部分得到了恢复。在补充有单油酸甘油酯的培养基中进行的生长研究表明，MsDelta0220 和 ComMsDelta0220S111A 突变株在存在这种脂质的情况下生长的能力都受到了损害。此外，对 MsDelta0220 菌株的抗微生物敏感性研究表明，该突变株比野生型菌株对利福平更敏感，对异烟肼更耐药，这表明该酶除了在水解外源性脂质方面的功能外，在分枝杆菌生理学中具有关键的结构作用。
