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Protein engineering of thromboxane synthase: conversion of membrane-bound to soluble form.

Abstract：

: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.

journal_name

Arch Biochem Biophys

journal_title

Archives of biochemistry and biophysics

authors

Hsu PY,Wang LH

doi

10.1016/s0003-9861(03)00287-x

subject

Has Abstract

pub_date

2003-08-01 00:00:00

pages

38-46

issue

1

eissn

0003-9861

issn

1096-0384

pii

S000398610300287X

journal_volume

416

pub_type

杂志文章

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