2,2‘,3,3‘,6,6‘-Hexachlorobiphenyl Hydroxylation by Active Site Mutants of Cytochrome P450 2B1 and 2B11

Abstract

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.

Chiral polychlorinated biphenyls: absorption, metabolism and excretion—a review

“…Species differences in the metabolism rates and metabolite profiles have been observed in many studies investigating the P450 enzyme-mediated oxidation of C-PCBs to HO-PCBs (Ohta et al 2013, Ohta et al 2005, Waller et al 1999, Warner et al 2009, Wu et al 2014). Several factors contribute to these differences, for example species-differences in the structure of relevant P450 enzymes (Waller et al 1999) and the hepatic P450 enzyme and P450 isoform composition (Lewis et al 1998).…”

“…A convenient nomenclature for PCB metabolites has been proposed by Maervoet et al and, as shown for PCB 136 in Fig 3, will be used throughout this manuscript (Maervoet et al 2004). The oxidation of C-PCBs, in particular those with a 2,3,6-trichloro substitution pattern in one phenyl ring, to HO-PCBs has been studied extensively using recombinant enzymes (Lu et al 2013, Lu and Wong 2011, Waller et al 1999, Warner et al 2009), hepatic microsomes (Kania-Korwel et al 2011, Kania-Korwel and Lehmler 2013, Schnellmann et al 1983, Wu et al 2014, Wu et al 2011), isolated hepatocytes (Vickers et al 1986) and, liver, hippocampus and skin slices (Garner et al 2006, Wu et al 2013a, Wu et al 2013b) obtained from mammalian species. To the best of our knowledge, the oxidation of C-PCBs in non-mammalian species, such as amphibians, fish or avian species, has not been investigated to date.…”

“…In addition, a 1,2-shift (or NIH-shift) (Guroff et al 1967, Jerina and Daly 1974) of the 3-chloro substituent can lead to the formation of HO-PCBs with a 2,4,6-trichloro-3-hydroxy substitution pattern as minor metabolites (Kania-Korwel et al 2012, Kania-Korwel et al 2011, Wu et al 2013a, Wu et al 2014). Studies with recombinant P450 enzymes demonstrate that CYP2B isoforms, such as rat CYP2B1 (Lu et al 2013, Waller et al 1999, Warner et al 2009), human CYP2B6 (Warner et al. 2009) and dog CYP2B11 (Waller et al 1999), are involved in the formation of meta hydroxylated C-PCBs.…”

“…Studies with recombinant P450 enzymes demonstrate that CYP2B isoforms, such as rat CYP2B1 (Lu et al 2013, Waller et al 1999, Warner et al 2009), human CYP2B6 (Warner et al. 2009) and dog CYP2B11 (Waller et al 1999), are involved in the formation of meta hydroxylated C-PCBs. The P450 isoforms responsible for the formation of para hydroxylated metabolites of C-PCBs have not been identified to date.…”

“…Several factors contribute to these differences, for example species-differences in the structure of relevant P450 enzymes (Waller et al 1999) and the hepatic P450 enzyme and P450 isoform composition (Lewis et al 1998). Studies with liver microsomes reveal that 5-HO-PCB 136 is formed by liver microsomes with a rank order of rat > dog ~ guinea pig ~ hamster > human > monkey ~ mouse ~ rabbit (Fig.…”