Determination of the structure of seleno-methionine-labelled hydroxymethylbilane synthase in its active form by multi-wavelength anomalous dispersion

The enzyme hydroxymethylbilane synthase (HMBS, E.C. 4.3.1.8) catalyzes the conversion of porphobilinogen into hydroxymethylbilane, a key intermediate for the biosynthesis of heme, chlorophylls, vitamin Bit and related macrocy cles. The enzyme is found in all organisms, except viruses. The crystal str ucture of the selenomethionine-labelled enzyme ([SeMet]HMBS) from Escherich ia coli has been solved by the multi-wavelength anomalous dispersion (MAD) experimental method using the Daresbury SRS station 9.5. In addition, [SeMe t]HMBS has been studied by MAD at the Grenoble ESRF MAD beamline BM14 (BL19 ) and this work is described especially with respect to the use of the ESRF CCD detector. The structure at ambient temperature has been refined, the R factor being 16.8% at 2.4 Angstrom resolution The dipyrromethane cofactor of the enzyme is preserved in its reduced form in the crystal and its geome trical shape is in full agreement with the crystal structures of authentic dipyrromethanes. Proximal to the reactive C atom of the reduced cofactor, s pherical density is seen consistent with there being a water molecule ideal ly placed to take part in the final step of the enzyme reaction cycle. Intr iguingly, the loop with residues 47-58 is not ordered in the structure of t his form of the enzyme, which carries no substrate. Direct experimental stu dy of the active enzyme is now feasible using time-resolved Laue diffractio n and freeze-trapping, building on the structural work described here as th e foundation.