Stohl E A, Brady S F, Clardy J, Handelsman J
Department of Plant Pathology, University of Wisconsin, Madison, Wisconsin 53706, USA.
J Bacteriol. 1999 Sep;181(17):5455-60. doi: 10.1128/JB.181.17.5455-5460.1999.
ZmaR is a resistance determinant of unusual abundance in the environment and confers on gram-positive and gram-negative bacteria resistance to zwittermicin A, a novel broad-spectrum antibiotic produced by species of Bacillus. The ZmaR protein has no sequence similarity to proteins of known function; thus, the purpose of the present study was to determine the function of ZmaR in vitro. Cell extracts of E. coli containing zmaR inactivated zwittermicin A by covalent modification. Chemical analysis of inactivated zwittermicin A by 1H NMR, 13C NMR, and high- and low-resolution mass spectrometry demonstrated that the inactivated zwittermicin A was acetylated. Purified ZmaR protein inactivated zwittermicin A, and biochemical assays for acetyltransferase activity with [14C]acetyl coenzyme A demonstrated that ZmaR catalyzes the acetylation of zwittermicin A with acetyl coenzyme A as a donor group, suggesting that ZmaR may constitute a new class of acetyltransferases. Our results allow us to assign a biochemical function to a resistance protein that has no sequence similarity to proteins of known function, contributing fundamental knowledge to the fields of antibiotic resistance and protein function.
ZmaR是一种在环境中含量异常丰富的抗性决定因子，它赋予革兰氏阳性菌和革兰氏阴性菌对双效霉素A的抗性，双效霉素A是一种由芽孢杆菌属产生的新型广谱抗生素。ZmaR蛋白与已知功能的蛋白质没有序列相似性；因此，本研究的目的是在体外确定ZmaR的功能。含有失活zmaR的大肠杆菌细胞提取物通过共价修饰使双效霉素A失活。通过1H NMR、13C NMR以及高分辨率和低分辨率质谱对失活的双效霉素A进行化学分析，结果表明失活的双效霉素A被乙酰化了。纯化的ZmaR蛋白使双效霉素A失活，并且用[14C]乙酰辅酶A进行的乙酰转移酶活性生化测定表明，ZmaR以乙酰辅酶A作为供体基团催化双效霉素A的乙酰化反应，这表明ZmaR可能构成一类新的乙酰转移酶。我们的结果使我们能够为一种与已知功能蛋白质没有序列相似性的抗性蛋白赋予生化功能，为抗生素抗性和蛋白质功能领域提供了基础知识。
