Protein engineering of thromboxane synthase: conversion of membrane-bound to soluble form.
Thromboxane A2 synthase (TXAS) binds to the endoplasmic reticulum membrane and catalyzes both an isomerization of prostaglandin H2 (PGH2) to form thromboxane A2 (TXA2) and a fragmentation of PGH2 to form 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and malondialdehyde (MDA). TXAS is a non-classic cytochrome P450 in that it does not require molecular oxygen or an external electron donor for catalysis. Difficulty in obtaining crystals from the membrane-bound TXAS prompted us to modify the protein to a soluble form. Results from site-directed mutagenesis, hydropathy analysis, and homology modeling led us to identify a putative membrane association segment near the end of helix F in TXAS. We report here the generation of a soluble form of TXAS by deletion of the amino-terminal membrane-anchoring domain and replacement of the helix F and F-G loop region with the corresponding region of the structurally characterized microsomal P450 2C5. The resultant TXAS/2C5 chimera is expressed in bacteria as a cytosolic and monomeric protein. Addition of an amino-terminal leader sequence to enhance expression and a tetra-histidine segment at the carboxyl-terminus to facilitate purification yielded approximately 4 mg of nearly homogeneous TXAS/2C5 per liter of bacterial culture. The TXAS/2C5 chimera contains heme at nearly a 1:1 molar ratio and catalyzes the formation of TXA2, MDA, and HHT at a 1:1:1 ratio, although with a reduced catalytic activity compared to wild type TXAS. TXAS/2C5 exhibits electronic absorption spectra similar to wild type TXAS and has similar affinities toward distal heme ligands such as imidazole and U44069. The chimera was mono-dispersive and thus is promising for crystallization trials.
Hsu
Pei-Yung
PY
Division of Hematology, Department of Internal Medicine, University of Texas-Houston, 6431 Fannin, Houston, TX 77030, USA.
Wang
Lee-Ho
LH
D000595:Amino Acid Sequence; D015870:Gene Expression; D006418:Heme / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011450:Prostaglandin Endoperoxides, Synthetic / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013930:Thromboxane-A Synthase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D007093:Imidazoles; D011450:Prostaglandin Endoperoxides, Synthetic; D011994:Recombinant Proteins; D006418:Heme; C028429:U 44069; C029899:imidazole; D013930:Thromboxane-A Synthase