Expression, Purification, and Spectroscopic Characterization of Human Thromboxane Synthase

Abstract

Thromboxane A 2 (TXA 2 ) is a potent inducer of vasoconstriction and platelet aggregation. Large scale expression of TXA 2 synthase (TXAS) is very useful for studies of the reaction mechanism, structural/functional relationships, and drug interactions. We report here a heterologous system for overexpression of human TXAS. The TXAS cDNA was modified by replacing the sequence encoding the first 28 amino acid residues with a CYP17 amino-terminal sequence and by adding a polyhistidine tag sequence prior to the stop codon; the cDNA was inserted into the pCW vector and co-expressed with chaperonins groES and groEL in Escherichia coli. The resulting recombinant protein was purified to electrophoretic homogeneity by affinity, ion exchange, and hydrophobic chromatography. UV-visible absorbance (UVVis), magnetic circular dichroism (MCD), and electron paramagnetic resonance (EPR) spectra indicate that TXAS has a typical low spin cytochrome P450 heme with an oxygen-based distal ligand. The UV-Vis and EPR spectra of recombinant TXAS were essentially identical to those of TXAS isolated from human platelets, except that a more homogenous EPR spectrum was observed for the recombinant TXAS. The recombinant protein had a heme:protein molar ratio of 0.7:1 and a specific activity of 12 mol of TXA 2 /min/mg of protein at 23°C. Furthermore, it catalyzed formation of TXA 2 , 12-hydroxy-5,8,10-heptadecatrienoic acid, and malondialdehyde in a molar ratio of 0.94:1.0:0.93. Spectral binding titrations showed that bulky heme ligands such as clotrimazole bound strongly to TXAS (K d ϳ0.5 M), indicating ample space at the distal face of the heme iron. Analysis of MCD and EPR spectra showed that TXAS was a typical low spin hemoprotein with a proximal thiolate ligand and had a very hydrophobic distal ligand binding domain.

Reaction mechanisms of 15-hydroperoxyeicosatetraenoic acid catalyzed by human prostacyclin and thromboxane synthases

“…We typically obtained 5 mg of purified TXAS per liter of culture medium (Table I). This yield of the hydrophilic N-terminal TXAS considerably exceeded that of TXAS with the hydrophobic Nterminus (∼0.8 mg/L of culture) [14].…”

“…We typically obtained 5 mg of purified TXAS per liter of culture medium (Table I). This yield of the hydrophilic N-terminal TXAS considerably exceeded that of TXAS with the hydrophobic Nterminus (∼0.8 mg/L of culture) [14].The UV-visible absorption spectrum of hydrophilic N-terminal TXAS is indistinguishable from that of hydrophobic N-terminal TXAS. The TXAS with hydrophilic N-terminus converts PGH 2 to TXA 2 , malondialdehyde and 12-hydroxy-5, 8, 10-heptadecatrienoic acid at a molar ratio of 1:1:1, with a turnover number, k cat , of 5,000 ± 300 min −1 and K M (PGH 2 ) of 29 ± 5 μM, comparable to the values for TXAS with hydrophobic N-terminus (k cat of 4,000 ± 300 min −1 ; K M , 21 ± 2 μM [20]).…”

“…The TXAS plasmid was transformed in E. coli BL21(DE3)pLysS, and the recombinant protein was induced by IPTG in the presence of δ-aminolevulinic acid. Purification of TXAS was modified from previous procedures [14]. Briefly, the frozen cells were re-suspended in 50 mM NaPi, pH 7.5 containing 0.1 M NaCl, 10% glycerol, 50 μg/ml DNase and 4 mM MgCl 2 , and then subjected to sonication.…”

“…TXAS then was eluted with 200 mM NaPi, pH 7.5 and 10% glycerol. The concentration of TXAS was determined using ε 418 = 100 mM −1 cm −1 [14]. Both PGIS and TXAS were stored at −70°C.…”

“…Furthermore, the residues from five substrate recognition sites in PGIS make up a highly hydrophobic environment with only two residues being hydrophilic [31]. "Truth diagram" analysis of heme EPR data indicates that both PGIS and TXAS have similar hydrophobicity in the distal heme pocket [13,14]; thus, the different volume of the heme pockets in both enzymes may be a more critical determinant for the product profile. As compared to other microsomal P450s, PGIS and TXAS take significant parts in the heterolytic cleavage, a feature of peroxidase reaction.…”