Cloning and characterization of a microsomal epoxide hydrolase from Heliothis virescens

Abstract

Epoxide hydrolases (EHs) are α/β-hydrolase fold superfamily enzymes that convert epoxides to 1,2-trans diols. In insects EHs play critical roles in the metabolism of toxic compounds and allelochemicals found in the diet and for the regulation of endogenous juvenile hormones (JHs). In this study we obtained a full-length cDNA, hvmeh1, from the generalist feeder Heliothis virescens that encoded a highly active EH, Hv-mEH1. Of the 10 different EH substrates that were tested, Hv-mEH1 showed the highest specific activity (1180 nmol min(-1) mg(-1)) for a 1,2-disubstituted epoxide-containing fluorescent substrate. This specific activity was more than 25- and 3900-fold higher than that for the general EH substrates cis-stilbene oxide and trans-stilbene oxide, respectively. Although phylogenetic analysis placed Hv-mEH1 in a clade with some lepidopteran JH metabolizing EHs (JHEHs), JH III was a relatively poor substrate for Hv-mEH1. Hv-mEH1 showed a unique substrate selectivity profile for the substrates tested in comparison to those of MsJHEH, a well-characterized JHEH from Manduca sexta, and hmEH, a human microsomal EH. Hv-mEH1 also showed unique enzyme inhibition profiles to JH-like urea, JH-like secondary amide, JH-like primary amide, and non-JH-like primary amide compounds in comparison to MsJHEH and hmEH. Although Hv-mEH1 is capable of metabolizing JH III, our findings suggest that this enzymatic activity does not play a significant role in the metabolism of JH in the caterpillar. The ability of Hv-mEH1 to rapidly hydrolyze 1,2-disubstituted epoxides suggests that it may play roles in the metabolism of fatty acid epoxides such as those that are commonly found in the diet of Heliothis.

Fig. 1

Structure of juvenile hormone (JH) III, JH II, JH I, and JH 0. At least seven forms of JH have been isolated from insects. All of the JHs that have been identified to date have an epoxide and a methyl ester.

Fig. 2

Nucleotide (lower case text) and amino acid (upper case text) sequences of hvmeh1 and Hv-mEH1, respectively. The 5′- and 3′-UTR sequences, and coding sequence of hvmeh1 were 53, 38, and 1,389 nts-long, respectively. Amino acid residues that form the putative catalytic triad (D-227, H-430, and E-403), lid domain (Y-298 and Y-373), and oxyanion hole (HGWP, residues 152–155) are shown in bold text. The asterisk indicates a stop codon (TGA). A putative membrane anchor domain (residues 2–24) that was predicted by SOSUI version 1.11 (Hirokawa et al., 1998) is shown in italic text. Amino acid residue positions are indicated to the right.

Fig. 3

Phylogenetic relatedness of Hv-mEH1 and JHEH or microsomal EH sequences from five insect orders and Primates. The phylogenetic analysis was performed using MEGA version 5.05 (Tamura et al., 2011). The tree was generated by the Neighbor-Joining method using a ClustalW generated alignment of 21 EH sequences. The percentage of replicate trees in which the sequences clustered together in the bootstrap analysis (1000 replicates) is shown at the branch nodes. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances (computed using the Poisson correction method) used to infer the phylogenetic tree. The GenBank accession number of each sequence is shown within the parenthesis. The insect order and reference of the sequences are as follows. Coleoptera: TcJHEH-r1, -r2, -r3, -r4, and -r5 (Tsubota et al., 2010); Diptera: DmEH (Taniai et al., 2003); Siphonaptera: CfEH1, CfEH2 (Keiser et al., 2002); Hymenoptera: Nasvi-EH1 (Abdel-Latief et al., 2008), AmJHEH (Mackert et al., 2010); Lepidoptera: BmJHEH, BmJHEH-r1, -r2, -r3, -r4, -r5 (Seino et al., 2010), Bommo-JHEH (Zhang et al., 2005), MsJHEH (Wojtasek and Prestwich, 1996), TmEH-1 (Harris et al., 1999); Primates: hmEH (Jackson et al., 1987).

Fig. 4

Effect of pH on the specific activity of Hv-mEH1 for JH III (A) and on non-enzymatic background hydrolysis of JH III (B). The activities were measured in citrate-phosphate (pH 4.0 and 5.0), sodium phosphate (pH 6.0, 7.0, and 8.0) and glycine-sodium hydroxide (pH 9.0 and 10.0) buffer containing 5 μM JH III and 0.1 mg ml⁻¹ of BSA. The specific activity values for Hv-mEH1 are corrected for background hydrolysis. The error bars indicate the standard deviation of the mean of at least three independent experiments.