Directed evolution reveals requisite sequence elements in the functional expression of P450 2F1 in Escherichia coli.
Cytochrome P450 2F1 (P450 2F1) is expressed exclusively in the human respiratory tract and is implicated in 3-methylindole (3MI)-induced pneumotoxicity via dehydrogenation of 3MI to a reactive electrophilic intermediate, 3-methyleneindolenine (3-MEI). Studies of P450 2F1 to date have been limited by the failure to express this enzyme in Escherichia coli. By contrast, P450 2F3, a caprine homologue that shares 84% sequence identity with P450 2F1 (86 amino acid differences), has been expressed in E. coli at yields greater than 250 nmol/L culture. We hypothesized that a limited number of sequence differences between P450s 2F1 and 2F3 could limit P450 2F1 expression in E. coli and that problematic P450 2F1 sequence elements could be identified by directed evolution. A library of P450 2F1/2F3 mutants was created by DNA family shuffling and screened for expression in E. coli. Three generations of DNA shuffling revealed a mutant (named JH_2F_F3_1_007) with 96.5% nucleotide sequence identity to P450 2F1 and which expressed 119 ± 40 pmol (n = 3, mean ± SD) hemoprotein in 1 mL microaerobic cultures. Across all three generations, two regions were observed where P450 2F3-derived sequence was consistently substituted for P450 2F1 sequence in expressing mutants, encoding nine amino acid differences between P450s 2F1 and 2F3: nucleotides 191-278 (amino acids 65-92) and 794-924 (amino acids 265-305). Chimeras constructed to specifically test the importance of these two regions confirmed that P450 2F3 sequence is essential in both regions for expression in E. coli but that other non-P450 2F1 sequence elements outside of these regions also improved the expression of mutant JH_2F_F3_1_007. Mutant JH_2F_F3_1_007 catalyzed the dehydrogenation of 3MI to 3-MEI as indicated by the observation of glutathione adducts after incubation in the presence of glutathione. The JH_2F_F3_1_007 protein differs from P450 2F1 at only 20 amino acids and should facilitate further studies of the structure-activity relationships of P450s of the 2F subfamily.
School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Brisbane 4072, Australia.
James B Y H
JB
Behrendorff
Chad D
CD
Moore
Keon-Hee
KH
Kim
Dae-Hwan
DH
Kim
Christopher A
CA
Smith
Wayne A
WA
Johnston
Chul-Ho
CH
Yun
Garold S
GS
Yost
Elizabeth M J
EM
Gillam
D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D007211:Indoles / Q000737:chemistry; D013058:Mass Spectrometry; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012862:Skatole / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
D007211:Indoles; D011994:Recombinant Proteins; C097745:3-methyleneindolenine; D003577:Cytochrome P-450 Enzyme System; D012862:Skatole; D005978:Glutathione