Purification, characterization, crystallisation and X-ray analysis of selenomethionine-labelled hydroxymethylbilane...

Hydroxymethylbilane synthase (HMBS) catalyses the conversion of porphobilinogen into hydroxymethylbilane, a linear tetrapyrrolic intermediate in the biosynthesis of haems, chlorophylls, vitamin B12 and related macrocycles. In the course of an investigation of the crystal structure of this enzyme, we intended to follow a new strategy to obtain the X-ray phase information, i.e. the collection of multiwavelength anomalous diffraction data from a crystal of a seleno-L-methionine (SeMet)-labelled variant of the protein. We have expressed and purified HMBS from Escherichia coli (34268 Da) in which all (six) methionine (Met) residues are replaced by SeMet. Complete replacement, as shown by amino acid composition analysis and by electrospray mass spectrometry, was achieved by growing the Met-requiring mutant E. coli PO1562 carrying the plasmid pPA410 in a medium containing 50 mg/l SeMet as the sole source of Met. [SeMet]HMBS exhibits full enzyme activity, as reflected by unchanged steady-state kinetic parameters relative to native enzyme. Rhombohedral crystals of [SeMet]HMBS could be grown at the pH optimum (7.4) of the enzyme (solutions containing 30 mg/ml protein, 0.4 mM EDTA, 20 mM dithiothreitol, 3 M NaCl and 15 mM Bristris-propane buffer were equilibrated by vapour diffusion at 20 degrees C against reservoirs of saturated NaCl). However, being very thin plates, these crystals were not suitable for X-ray analysis. Alternatively, rectangular crystals were obtained at pH 5.3 using conditions based on those reported for wild-type HMBS [sitting drops of 50 microliters containing 6-7 mg/ml protein, 0.3 mM EDTA, 15 mM dithiothreitol, 10% (mass/vol.) poly(ethylene glycol) 6000 and 0.01% NaN3 in 0.1 M sodium acetate were equilibrated by vapour diffusion at 20 degrees C against a reservoir of 10-20 mg solid dithiothreitol]. X-ray diffraction data of the crystals were complete to 93.8% at 0.21 nm resolution and showed that [SeMet]HMBS and native HMBS crystallise isomorphously. A difference Fourier map using FSeMet-Fnative and phases derived from the native structure, which has recently been determined independently by multiple isomorphous replacement, showed positive difference peaks centered at or close to where the sulphur atoms of the Met side chains appear in the native structure. In addition, paired positive/negative peaks in the difference map near the cofactor of HMBS indicate conformational differences in the active site, probably due to differences in the state of oxidation of the cofactor in the two crystalline samples.