摘要
[目的]本文旨在研究土壤宏基因组来源的新型四环素破坏酶Tet(64)的耐药机制并分析tet(64)的基因环境,为tet(64)基因的传播及抗生素和佐剂的研发奠定基础。[方法]利用体外生化反应验证Tet(64)降解四环素的功能,通过液相色谱-质谱联用(LC-MS)分析降解产物,结合序列比对、系统发育树构建、分子对接等生物信息学方法推测Tet(64)中与四环素结合的重要氨基酸残基,解析Tet(64)的耐药机制和tet(64)基因的水平转移风险。[结果]MQ776克隆的序列分析发现tet(64)上游存在IS1插入序列,暗示tet(64)基因具有潜在的水平转移风险。Tet(64)属于黄素依赖型单加氧酶,与首次发现的四环素破坏酶Tet(X)仅有18.93%的序列一致性,与最近发现的一系列土壤来源四环素破坏酶Tet(47)—Tet(56)也只有70%左右的序列一致性。SWISS-MODEL三维建模发现Tet(64)与土壤来源的Tet(50)具有相似的结构,包含1个四环素结合域、1个FAD结合域以及连接上述2个结构域的2个α-螺旋。体外生化试验发现Tet(64)降解四环素后产生了2个特异产物,m/z均为461.0,与Tet(X)催化产物一致。推测反应中四环素C11a位点被羟基化,破坏了C11和C12组成的β-二酮区域,导致四环素不能螯合镁离子而失去抗菌活性。[结论]鉴定了土壤来源的新型四环素破坏酶Tet(64)的四环素降解功能,初步解析了Tet(64)的耐药机制。
[Objectives]This paper aimed to study the resistance mechanism of metagenome derived tetracycline destructase Tet(64)and analyze the gene environment of tet(64)in order to lay a foundation for monitoring the transmission of tetracycline-destroying enzyme genes and the development of antibiotics and adjuvants.[Methods]The degradation activity of Tet(64)on tetracycline was confirmed by biochemical reaction in vitro,and then the degradation products were analyzed by liquid chromatograph-mass spectrometer(LC-MS)to elucidate the resistance mechanism of the tetracycline destructase.The key amino acid residues of Tet(64)binding to tetracycline and the possibility of horizontal transfer of tet(64)were discussed by bioinformatics including multiple sequence alignment,phylogenetic tree construction,and molecular docking methods.[Results]Sequence analysis of MQ776 clone revealed that two IS1 insertions were located upstream of tet(64),implying the potential transfer risk of the tetracycline destructase gene.Tet(64)was a flavin-dependent monooxygenase,and shared identities of 18.93% and approximate 70% with the first discovered tetracycline destructase Tet(X)and soil-derived tetracycline destructases Tet(47)-Tet(56),respectively.The predicted 3D structure modeled by SWISS-MODEL revealed that Tet(64)had a similar structure with the soil-derived tetracycline destructase Tet(50),which contained a tetracycline-binding domain,a FAD-binding domain,and twoα-helices.Tet(64)degraded tetracycline into two products with m/z of 461.0 in vitro,which was same as that of Tet(X).Thus,it was implied that Tet(64)hydroxylated the C11a site of tetracycline to destroy theβ-diketone moiety(C11 and C12),resulting in the failure of chelating magnesium ions and the loss of antibacterial activity.[Conclusions]The degradation activity of the tetracycline destructase Tet(64)on tetracycline was confirmed,and the resistance mechanism of Tet(64)was preliminarily analyzed.
作者
荣宇凤
王绍琛
卫林
高月皎
吕云斌
冯治洋
RONG Yufeng;WANG Shaochen;WEI Lin;GAO Yuejiao;L Yunbin;FENG Zhiyang(College of Food Science and Technology,Nanjing Agricultural University,Nanjing 210095,China)
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2023年第2期387-395,共9页
Journal of Nanjing Agricultural University
基金
国家自然科学基金项目(31770049)。
关键词
四环素破坏酶
生化分析
耐药机制
分子对接
tetracycline destructase
biochemical analysis
resistance mechanism
molecular docking
