Waller S C, He Y A, Harlow G R, He Y Q, Mash E A, Halpert J R
Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77555-1031, USA.
Chem Res Toxicol. 1999 Aug;12(8):690-9. doi: 10.1021/tx990030j.
The structural basis of species differences in cytochrome P450 2B-mediated hydroxylation of 2,2',3,3',6,6'-hexachlorobiphenyl (236HCB) was evaluated by using 14 site-directed mutants of cytochrome P450 2B1 and three point mutants of 2B11 expressed in Escherichia coli. To facilitate metabolite identification, seven possible products, including three hydroxylated and four dihydroxylated hexachlorobiphenyls, were synthesized by direct functionalization of precursors and Ullmann and crossed Ullmann reactions. HPLC and GC/MS analysis and comparison with authentic standards revealed that 2B1, 2B11, and all their mutants produced 4, 5-dihydroxy-236HCB and 5-hydroxy-236HCB, while 2B11 L363V and 2B1 I114V mutants also catalyzed hydroxylation at the 4-position. The amount of products formed by 2B1 mutants I114V, F206L, L209A, T302S, V363A, V363L, V367A, I477A, I477L, G478S, I480A, and I480L was smaller than that of the wild type. I477V exhibited unaltered 236HCB metabolism, and I480V produced twice as much dihydroxy product as the wild type. For 2B11, substitution of Val-114 or Asp-290 with Ile decreased the product yields. Replacement of Leu-363 with Val dramatically altered the profile of 236HCB metabolites. In addition to an increase in the overall level of hydroxylation, the mutant mainly catalyzed hydroxylation at the 4-position. Incubation of P450 2B1 with 5-hydroxy-236HCB produced 4,5-dihydroxy-236HCB, which indicates that 4,5-dihydroxy-236HCB may be formed by a direct hydroxylation of 5-hydroxy-236HCB. The findings from this study demonstrate the importance of residues 114, 206, 209, 302, 363, 367, 477, 478, and 480 in 2B1 and 114, 290, and 363 in 2B11 for 236HCB metabolism.
通过使用在大肠杆菌中表达的细胞色素P450 2B1的14个定点突变体和2B11的3个点突变体，评估了细胞色素P450 2B介导的2,2',3,3',6,6'-六氯联苯（236HCB）羟基化物种差异的结构基础。为便于代谢物鉴定，通过前体的直接官能化以及乌尔曼反应和交叉乌尔曼反应合成了7种可能的产物，包括3种羟基化和4种二羟基化的六氯联苯。HPLC和GC/MS分析以及与标准品的比较表明，2B1、2B11及其所有突变体均产生4,5-二羟基-236HCB和5-羟基-236HCB，而2B11 L363V和2B1 I114V突变体还催化4位的羟基化。2B1突变体I114V、F206L、L209A、T302S、V363A、V363L、V367A、I477A、I477L、G478S、I480A和I480L形成的产物量比野生型少。I477V的236HCB代谢未改变，I480V产生的二羟基产物是野生型的两倍。对于2B11，用异亮氨酸取代Val-114或Asp-290会降低产物产率。用缬氨酸取代Leu-363显著改变了236HCB代谢物的谱。除了羟基化总体水平增加外，该突变体主要催化4位的羟基化。细胞色素P450 2B1与5-羟基-236HCB孵育产生4,5-二羟基-236HCB，这表明4,5-二羟基-236HCB可能由5-羟基-236HCB的直接羟基化形成。本研究结果证明了2B1中的114、206、209、302、363、367、477、478和480位以及2B11中的114、290和363位残基对236HCB代谢的重要性。
