Purification, characterization, crystallisation and X‐ray analysis of selenomethionine‐labelled hydroxymethylbilane synthase from Escherichia coli

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Abstract
Hydroxymethylbilane synthase (HMBS) catalyses the conversion of porphobilinogen into hydroxymethylbilane, a linear tetrapyrrolic intermediate in the biosynthesis of haems, chlorophylls, vitamin B12 and related macrocyles. 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 Bistris‐propane buffer were equilibrated by vapour diffusion at 20°C against reservours of saturated NaCl); However being very thin plates, these crystals were not suitable for X‐ray analysis. Alternatively, rectangular craystals were obtained at pH 5.3 using conditions based on those reported for wile‐type HMBS [sitting drops of 50 μl containing 6–7 mg/ml protein, 0.3, < 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°C aginst a reservoir of 10–20 mg solid dithiothreitol]. X‐ray difracion data of the crystals were complete to 93.8% at 0.21 nm resolution and showed that [SeMet]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 replacemetn, showed positive difference peaks centered at or closeto where the sulphur atmos of the Met side chains apper in the native structure. In addition, paird positive/negative peaks in the difference map near the cofactor of HMBS indicate conformational difference in the active site, probable dir to diffrences in the state of oxidaion of the cofactor in two crystalline samples.

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