Mosher R H, Camp D J, Yang K, Brown M P, Shaw W V, Vining L C
Biology Department, Dalhousie University, Halifax, Nova Scotia, Canada.
J Biol Chem. 1995 Nov 10;270(45):27000-6. doi: 10.1074/jbc.270.45.27000.
Plasmid pJV4, containing a 2.4-kilobase pair insert of genomic DNA from the chloramphenicol (Cm) producer Streptomyces venezuelae ISP5230, confers resistance when introduced by transformation into the Cm-sensitive host Streptomyces lividans M252 (Mosher, R. H. Ranade, N. P., Schrempf, H., and Vining, L. C. (1990) J. Gen. Microbiol. 136, 293-301). Transformants rapidly metabolized Cm to one major product, which was isolated and purified by reversed phase chromatography. The metabolite was identified by nuclear magnetic resonance spectroscopy and mass spectrometry as 3'-O-phospho-Cm, and was shown to have negligible inhibitory activity against Cm-sensitive Micrococcus luteus. The nucleotide sequence of the S. venezuelae DNA insert in pJV4 contains an open reading frame (ORF) that encodes a polypeptide (19 kDa) with a consensus motif at its NH2 terminus corresponding to a nucleotide-binding amino acid sequence (motif A or P-loop; Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 1, 945-951). When a recombinant vector containing this ORF as a 1.6-kilobase pair SmaI-SmaI fragment was used to transform S. lividans M252, uniformly Cm-resistant transformants were obtained. A strain of S. lividans transformed by a vector in which the ORF had been disrupted by an internal deletion yielded clones that were unable to phosphorylate Cm, and exhibited normal susceptibility to the antibiotic. The results implicate the product of the ORF from S. venezuelae as an enzymic effector of Cm resistance in the producing organism by 3'-O-phosphorylation. We suggest the trivial name chloramphenicol 3'-O-phosphotransferase for the enzyme.
质粒pJV4含有来自氯霉素（Cm）产生菌委内瑞拉链霉菌ISP5230的2.4千碱基对基因组DNA插入片段，当通过转化导入对Cm敏感的宿主变铅青链霉菌M252时可赋予抗性（莫舍，R.H.、拉纳德，N.P.、施雷姆普夫，H.和维宁，L.C.（1990年）《普通微生物学杂志》136，293 - 301）。转化体迅速将Cm代谢为一种主要产物，该产物通过反相色谱法分离纯化。通过核磁共振光谱和质谱鉴定该代谢产物为3'-O-磷酸氯霉素，并显示其对Cm敏感的藤黄微球菌的抑制活性可忽略不计。pJV4中委内瑞拉链霉菌DNA插入片段的核苷酸序列包含一个开放阅读框（ORF），该开放阅读框编码一种多肽（19 kDa），其NH2末端具有一个与核苷酸结合氨基酸序列（基序A或P环；沃克，J.E.、萨拉斯特，M.、伦斯威克，M.J.和盖伊，N.J.（1982年）《欧洲分子生物学组织杂志》1，945 - 951）一致的基序。当使用含有作为1.6千碱基对SmaI - SmaI片段的该ORF的重组载体转化变铅青链霉菌M252时，获得了均匀抗Cm的转化体。用一个载体转化的变铅青链霉菌菌株，其中ORF因内部缺失而被破坏，产生的克隆无法使Cm磷酸化，并且对抗生素表现出正常的敏感性。结果表明，委内瑞拉链霉菌ORF的产物是产生菌中通过3'-O-磷酸化赋予Cm抗性的酶促效应物。我们建议将该酶简称为氯霉素3'-O-磷酸转移酶。
