Expression of rat microsomal epoxide hydrolase in Escherichia coli. Identification of a histidyl residue essential for catalysis

• Published in: The Journal of biological chemistry Vol. 268; no. 19; pp. 14011 - 14017
• Main Authors: BELL, P. A, KASPER, C. B
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 05-07-1993
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Binding Sites; Biological and medical sciences; Catalysis; Cloning, Molecular - methods; Enzymes and enzyme inhibitors; Epoxide Hydrolases - genetics; Epoxide Hydrolases - isolation & purification; Epoxide Hydrolases - metabolism; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Histidine; Humans; Hydrogen-Ion Concentration; Kinetics; Microsomes - enzymology; Molecular Sequence Data; Mutagenesis, Site-Directed; Plasmids; Rabbits; Rats; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Restriction Mapping; Sequence Homology, Amino Acid; Transferases; Vertebrata; Mammalia; Histidine; Enzymatic activity; Rat; Enzyme; Active site; Rodentia; pH; Kinetics; Molecular cloning; Epoxide hydrolase
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)85202-X

The cDNA containing the complete coding region for rat microsomal epoxide hydrolase (EC 3.3.2.3) was cloned into the expression/secretion
vector pIN-III-OmpA3 and expressed in Escherichia coli strain TG1. Recombinant epoxide hydrolase was found to represent 4-9%
of total bacterial protein and catalyzed the hydrolysis of styrene oxide and benzo[a]pyrene 4,5-oxide with specific activities
of 421 and 734 nmol min-1 mg of epoxide hydrolase-1, respectively. Previous work implicated a histidyl residue at or near
the active site of the enzyme (DuBois, G. C., Appella, E., Levin, W., Lu, A. Y. H., and Jerina, D. M. (1978) J. Biol. Chem.
253, 2932-2939). Comparison of the amino acid sequences of rat, human, and rabbit epoxide hydrolases revealed the presence
of 14 conserved histidyl residues. To investigate the role of these residues in epoxide hydrolysis, site-specific mutants
were generated and expressed in E. coli. Mutants H64L, H82L, H115N, H126N, H129L, H148N, H170L, H176L, H242L, H247L, H301L,
H385L, K386M-H387L, delta 385-391, and H407L catalyzed the hydrolysis of benzo[a]pyrene 4,5-oxide with specific activities
between 115 and 830 nmol min-1 mg-1. Mutants H431L, H431N, and H431R were all found to have activities of < 5 nmol min-1 mg-1,
which is at least 150-fold less than the activity of the wild type enzyme. A Vm versus pH profile for the recombinant wild
type epoxide hydrolase revealed a broad pH optimum of 6.5 to 8.5 and the presence of three ionizable groups with pKa values
of 5.8 +/- 0.2, 9.2 +/- 0.1, and 9.7 +/- 0.4. The group with a pKa of 5.8 is preferentially unprotonated, while the other
two groups are preferentially protonated for catalysis. We propose that histidine 431 corresponds to the group with a pKa
of 5.8, while the others, with pKa values of 9.2 and 9.7 likely represent lysyl, cysteinyl, or tyrosyl residues. Thus, the
data are consistent with a model where His-431 acts as a general base, abstracting a proton from water, while another residue(s),
perhaps lysine, act as a general acid protonating the alkoxide anion that forms upon cleavage of the carbon-oxygen bond.