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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.

The role of conserved arginine residues in hydroxymethylbilane synthase was investigated by replacing these residues in the enzyme from Escherichia coli with leucine residues by using site-directed mutagenesis. The kinetic parameters for these mutant enzymes and studies on the formation of intermediate enzyme-substrate complexes indicate that several of these arginine residues are involved in binding the carboxylate side chains of the pyrromethane cofactor and the growing oligopyrrole chain.

A new construct carrying the hemC gene was transformed into Escherichia coli, resulting in approx. 1000-fold over-expression of hydroxymethylbilane synthase (HMBS). This construct was used to generate HMBS in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 were replaced by glutamine (K55Q, K59Q and K55Q-K59Q respectively). All three modified enzymes are chromatographically separable from wild-type enzyme. Kinetic studies showed that the substitution K55Q has little effect whereas K59Q causes a 25-fold decrease in Kapp. cat./Kapp. m. Treatment of K55Q, K59Q and K55Q-K59Q separately with pyridoxal 5'-phosphate and NaBH4 resulted in incomplete and non-specific reaction with the remaining lysine residues. Pyridoxal modification of Lys-59 in the K55Q mutant caused greater enzymic inactivation than similar modification of Lys-55 in K59Q. The results in sum show that, though Lys-55 and Lys-59 may be at or near the active site, neither is indispensable for the catalytic activity of HMBS.

Hydroxymethylbilane synthase (porphobilinogen deaminase) was purified to apparent homogeneity from Escherichia coli. The enzyme is a monomer of Mr approx. 40,000. The Km for porphobilinogen and relative Vmax. values have been obtained at various pH values over the range 6.2-8.8, enabling pK values for ionizable groups important for activity to be determined. The N-terminal amino acid sequence is presented.