Characterization of LtsA from Rhodococcus erythropolis, an enzyme with glutamine amidotransferase activity
Characterization of LtsA from Rhodococcus erythropolis, an enzyme with glutamine amidotransferase activity
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DOI：
10.1128/jb.187.8.2582-2591.2005
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发表时间：
2005-04-01
影响因子：
3.2
通讯作者：
Tamura, T
中科院分区：
文献类型：
--
作者：
Mitani, Y;Meng, XY;Tamura, T
诺卡菌型放线菌红平红球菌具有特征性的细胞壁结构。细胞壁由阿拉伯半乳聚糖和分枝菌酸组成，对溶菌酶（胞壁酶）的细胞壁溶解活性具有高度抵抗力。为了改进从红平红平菌宿主细胞中分离重组蛋白（N. Nakashima 和 T. Tamura, Biotechnol. Bioeng. 86:136-148, 2004），我们分离了两种突变体，L-65和L-88，它们对溶菌酶处理敏感。突变体的溶菌酶敏感性通过谷氨酸棒杆菌ItsA的表达得到补充，其编码具有谷氨酰胺酰胺转移酶活性的酶，该活性是由两个反应（谷氨酰胺酶活性和合成酶活性）耦合产生的。突变体的溶菌酶敏感性也得到来自枯草芽孢杆菌和结核分枝杆菌的ItsA同源物的补充，但来自天蓝色链霉菌和大肠杆菌的同源物不能补充敏感性。这一结果表明，只有某些 LtsA 同源物才能赋予溶菌酶抗性。来自红平红球菌的野生型重组 LtsA 显示出谷氨酰胺酶活性，但来自 L-88 和 L-65 突变体的 LtsA 酶显示出显着降低的活性。有趣的是，当将NH4Cl添加到培养基中时，表达突变LtsA的ItsA破坏突变体从溶菌酶敏感变为溶菌酶抗性。通过定点诱变失活的LtsA突变体的谷氨酰胺酶活性也得到恢复。经过。添加NH4Cl表明NH3可以用作酰胺供体分子。总而言之，这些结果表明 LtsA 在介导红平红细胞中的溶菌酶抗性中发挥着重要作用。
The nocardioform actinomycete Rhodococcus erythropolis has a characteristic cell wall structure. The cell wall is composed of arabinogalactan and mycolic acid and is highly resistant to the cell wall-lytic activity of lysozyme (muramidase). In order to improve the isolation of recombinant proteins from R. erythropolis host cells (N. Nakashima and T. Tamura, Biotechnol. Bioeng. 86:136-148, 2004), we isolated two mutants, L-65 and L-88, which are susceptible to lysozyme treatment. The lysozyme sensitivity of the mutants was complemented by expression of Corynebacterium glutamicum ItsA, which codes for an enzyme with glutamine amidotransferase activity that results from coupling of two reactions (a glutaminase activity and a synthetase activity). The lysozyme sensitivity of the mutants was also complemented by ItsA homologues from Bacillus subtilis and Mycobacterium tuberculosis, but the homologues from Streptomyces coelicolor and Escherichia coli did not complement the sensitivity. This result suggests that only certain LtsA homologues can confer lysozyme resistance. Wild-type recombinant LtsA from R. erythropolis showed glutaminase activity, but the LtsA enzymes from the L-88 and L-65 mutants displayed drastically reduced activity. Interestingly, an ItsA disruptant mutant, which expressed the mutated LtsA, changed from lysozyme sensitive to lysozyme resistant when NH4Cl was added into the culture media. The glutaminase activity of the LtsA mutants inactivated by site-directed mutagenesis was also restored. by. addition of NH4Cl indicating that NH3 can be used as an amide donor molecule. Taken together, these results suggest that LtsA is critically involved in mediating lysozyme resistance in R. erythropolis cells.