Bhushan Bharat, Halasz Annamaria, Hawari Jalal
Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montreal, Que., Canada H4P 2R2.
Biochem Biophys Res Commun. 2004 Sep 10;322(1):271-6. doi: 10.1016/j.bbrc.2004.07.115.
Previously, we reported that a salicylate 1-monooxygenase from Pseudomonas sp. ATCC 29352 biotransformed CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaaza-isowurtzitane) (C(6)H(6)N(12)O(12)) and produced a key metabolite with mol. wt. 346 Da corresponding to an empirical formula of C(6)H(6)N(10)O(8) which spontaneously decomposed in aqueous medium to produce N(2)O, NH(4)(+), and HCOOH [Appl. Environ. Microbiol. (2004)]. In the present study, we found that nitroreductase from Escherichia coli catalyzed a one-electron transfer to CL-20 to form a radical anion (CL-20(-)) which upon initial N-denitration also produced metabolite C(6)H(6)N(10)O(8). The latter was tentatively identified as 1,4,5,8-tetranitro-1,3a,4,4a,5,7a,8,8a-octahydro-diimidazo[4,5-b:4',5'-e]pyrazine [IUPAC] which decomposed spontaneously in water to produce glyoxal (OHCCHO) and formic acid (HCOOH). The rates of CL-20 biotransformation under anaerobic and aerobic conditions were 3.4+/-0.2 and 0.25+/-0.01 nmol min(-1)mg of protein(-1), respectively. The product stoichiometry showed that each reacted CL-20 molecule produced about 1.8 nitrite ions, 3.3 molecules of nitrous oxide, 1.6 molecules of formic acid, 1.0 molecule of glyoxal, and 1.3 ammonium ions. Carbon and nitrogen products gave mass-balances of 60% and 81%, respectively. A comparative study between native-, deflavo-, and reconstituted-nitroreductase showed that FMN-site was possibly involved in the biotransformation of CL-20.
此前，我们报道过来自假单胞菌属ATCC 29352的水杨酸1-单加氧酶可对六硝基六氮杂异伍兹烷（CL-20，2,4,6,8,10,12-六硝基-2,4,6,8,10,12-六氮杂异伍兹烷）（C₆H₆N₁₂O₁₂）进行生物转化，并产生一种关键代谢物，其分子量为346 Da，对应经验式C₆H₆N₁₀O₈，该代谢物在水介质中自发分解生成一氧化二氮、铵离子和甲酸[《应用与环境微生物学》（2004年）]。在本研究中，我们发现大肠杆菌的硝基还原酶催化向CL-20进行单电子转移，形成自由基阴离子（CL-20⁻），该自由基阴离子在最初的N-脱硝反应中也生成代谢物C₆H₆N₁₀O₈。后者初步鉴定为1,4,5,8-四硝基-1,3a,4,4a,5,7a,8,8a-八氢-二咪唑并[4,5-b:4',5'-e]吡嗪[国际纯粹与应用化学联合会（IUPAC）命名]，其在水中自发分解生成乙二醛（OHCCHO）和甲酸（HCOOH）。厌氧和好氧条件下CL-20的生物转化速率分别为3.4±0.2和0.25±0.01 nmol min⁻¹mg蛋白⁻¹。产物化学计量关系表明，每个反应的CL-20分子产生约1.8个亚硝酸根离子、3.3个一氧化二氮分子、1.6个甲酸分子、1.0个乙二醛分子和1.3个铵离子。碳和氮产物的质量平衡分别为60%和81%。对天然、脱黄素和重组硝基还原酶的比较研究表明，黄素单核苷酸（FMN）位点可能参与了CL-20的生物转化。
