Expression, purification, crystallization and preliminary X-ray studies of a prolyl-4-hydroxylase protein from Bacillus anthracis

Abstract

Collagen prolyl-4-hydroxylase (C-P4H) catalyzes the hydroxylation of specific proline residues in procollagen, which is an essential step in collagen biosynthesis. A new form of P4H from Bacillus anthracis (anthrax-P4H) that shares many characteristics with the type I C-P4H from human has recently been characterized. The structure of anthrax-P4H could provide important insight into the chemistry of C-P4Hs and into the function of this unique homodimeric P4H. X-ray diffraction data of selenomethionine-labeled anthrax-P4H recombinantly expressed in Escherichia coli have been collected to 1.4 A resolution.

Figure 1

Reaction catalyzed by P4H.

Figure 2

Crystals of anthrax-P4H grown using the hanging-drop method in 16%(w/v) PEG 8000, 40 mM potassium phosphate (monobasic) and 20%(v/v) glycerol pH 4.0. The approximate dimensions of a typical crystal are 0.8 × 0.5 × 0.1 mm.

Figure 3

Diffraction pattern of SeMet-anthrax-P4H obtained on beamline BL9-2, SSRL. The resolution of this data set was 1.4 Å.