Priefert Horst, O'Brien Xian M, Lessard Philip A, Dexter Annette F, Choi Ellen E, Tomic Sladjana, Nagpal Geeta, Cho Jennie J, Agosto Melina, Yang Lucy, Treadway Sheri L, Tamashiro Lance, Wallace Matthew, Sinskey Anthony J
Department of Biology, 68-370, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Appl Microbiol Biotechnol. 2004 Aug;65(2):168-76. doi: 10.1007/s00253-004-1589-3. Epub 2004 Apr 7.
Rhodococcus sp. I24 can oxygenate indene via at least three independent enzyme activities: (i) a naphthalene inducible monooxygenase (ii) a naphthalene inducible dioxygenase, and (iii) a toluene inducible dioxygenase (TID). Pulsed field gel analysis revealed that the I24 strain harbors two megaplasmids of approximately 340 and approximately 50 kb. Rhodococcus sp. KY1, a derivative of the I24 strain, lacks the approximately 340 kb element as well as the TID activity. Southern blotting and sequence analysis of an indigogenic, I24-derived cosmid suggested that an operon encoding a TID resides on the approximately 340 kb element. Expression of the tid operon was induced by toluene but not by naphthalene. In contrast, naphthalene did induce expression of the nid operon, encoding the naphthalene dioxygenase in I24. Cell free protein extracts of Escherichia coli cells expressing tidABCD were used in HPLC-based enzyme assays to characterize the indene bioconversion of TID in vitro. In addition to 1-indenol, indene was transformed to cis-indandiol with an enantiomeric excess of 45.2% of cis-(1S,2R)-indandiol over cis-(1R,2S)-indandiol, as revealed by chiral HPLC analysis. The Km of TID for indene was 380 microM. The enzyme also dioxygenated naphthalene to cis-dihydronaphthalenediol with an activity of 78% compared to the formation of cis-indandiol from indene. The Km of TID for naphthalene was 28 microM. TID converted only trace amounts of toluene to 1,2-dihydro-3-methylcatechol after prolonged incubation time. The results indicate the role of the tid operon in the bioconversion of indene to 1-indenol and cis-(1S,2R)-indandiol by Rhodococcus sp. I24.
红球菌属菌株I24可通过至少三种独立的酶活性将茚氧化：（i）萘诱导型单加氧酶；（ii）萘诱导型双加氧酶；以及（iii）甲苯诱导型双加氧酶（TID）。脉冲场凝胶分析显示，I24菌株含有两个大小约为340 kb和50 kb的大质粒。红球菌属菌株KY1是I24菌株的衍生物，它缺乏约340 kb的元件以及TID活性。对一个产靛蓝的、源自I24的黏粒进行Southern印迹和序列分析表明，一个编码TID的操纵子位于约340 kb的元件上。tid操纵子的表达由甲苯诱导，而非萘。相反，萘确实诱导了I24中编码萘双加氧酶的nid操纵子的表达。利用表达tidABCD的大肠杆菌细胞的无细胞蛋白提取物，通过基于高效液相色谱的酶分析来体外表征TID对茚的生物转化。手性高效液相色谱分析表明，除了1-茚醇外，茚还被转化为顺式茚二醇，其中顺式-(1S,2R)-茚二醇对顺式-(1R,2S)-茚二醇的对映体过量为45.2%。TID对茚的Km值为380 μM。该酶还将萘双加氧化为顺式二氢萘二醇，与从茚形成顺式茚二醇相比，活性为78%。TID对萘的Km值为28 μM。长时间孵育后，TID仅将痕量的甲苯转化为1,2-二氢-3-甲基邻苯二酚。结果表明tid操纵子在红球菌属菌株I24将茚生物转化为1-茚醇和顺式-(1S,2R)-茚二醇中的作用。
