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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 B12 and related macrocycles. The enzyme is found in all organisms, except viruses. The crystal structure of the selenomethionine-labelled enzyme ([SeMet]HMBS) from Escherichia coli has been solved by the multi-wavelength anomalous dispersion (MAD) experimental method using the Daresbury SRS station 9.5. In addition, [SeMet]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 A resolution. The dipyrromethane cofactor of the enzyme is preserved in its reduced form in the crystal and its geometrical shape is in full agreement with the crystal structures of authentic dipyrromethanes. Proximal to the reactive C atom of the reduced cofactor, spherical density is seen consistent with there being a water molecule ideally placed to take part in the final step of the enzyme reaction cycle. Intriguingly, the loop with residues 47-58 is not ordered in the structure of this form of the enzyme, which carries no substrate. Direct experimental study of the active enzyme is now feasible using time-resolved Laue diffraction and freeze-trapping, building on the structural work described here as the foundation.

Hydroxymethylbilane synthase (HMBS) catalyses the conversion of porphobilinogen into hydroxymethylbilane, a linear tetrapyrrolic intermediate in the biosynthesis of haems, chlorophylls, vitamin B,, 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 50mg/l SeMet as the sole source of Met. [SeMetIHMBS exhibits full enzyme activity, as reflected by unchanged steady-state kinetic parameters relative to native enzyme. Rhombohedral crystals of [SeMetIHMBS could be grown at the pH optimum (7.4) of the enzyme (solutions containing 30 mg/ml protein, 0.4 mh4 EDTA, 20 mM dithiothreitol, 3 M NaCl and 15 mM Bistris-propane buffer were equilibrated by vapour diffusion at 20 "C against reservoirs of saturated NaC1). 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 p1 containing 6-7 mg/ml protein, 0.3 mh4 EDTA, 15 mM dithiothreitol, 10 % (mass/vol.) poly(ethy1ene glycol) 6000 and 0.01 % NaN, in 0.1 M sodium acetate were equilibrated by vapour diffusion at 20°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 [SeMetIHMBS 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 positivehegative 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 replacement of methionine residues by seleno-Lmethionine (SeMet) in proteins is part of a new strategy to tackle the cardinal problem of protein crystallography : the determination of phases [l]. Given the availability of welldiffracting crystals of a SeMet-containing protein, the ap- proach involves the collection of multiwavelength anomalous diffraction (MAD) data from a single crystal using synchrotron radiation. Specific algebraic methods for MAD data analysis allow the evaluation of phase angles for the diffracte...

(Hydroxymethy1)bilane synthase (HMBS) catalyses the conversion of porphobilinogen (2) into the (hydroxymethy1)bilane derivative 3, a linear tetrapyrrolic intermediate in the biosynthesis of haem, chlorophyll, and related pigments. The conversion involves the sequential formation of four intermediate covalent enzyme-substrate complexes, before the product is released. We analysed the pre-steady-state kinetics of the formation of the complexes, taking advantage of their remarkable chemical stability allowing chromatographic separation. The experimental approach involved the generation of the complexes while HMBS was immobilised on an anion-exchange column. A solution being 0.2 K,,, in substrate was pumped through the column during a time interval which was varied to sample the pre-steady-state period. Then, the enzyme and enzyme-substrate complexes were eluted and their proportions evaluated. A computer simulation of the pre-steady-state time course, in combination with a x 2 fitting to the experimental data, allowed the specificity constants k,,JK, for the individual steps of the process to be derived. By repeating the analysis with variants of HMBS in which specific amino acids were replaced by others, we demonstrated that it is possible to trace the consequences of amino-acid replacements down to the individual steps of the reaction sequence. Since the positions of the amino acids concerned in the three-dimensional structure were known, detailed structure-function relationships become evident in this way.Introduction. -(Hydroxymethy1)bilane synthase (HMBS, EC 4.3.1.8, also known as porphobilinogen deaminase) is an enzyme of the biosynthetic pathway leading to haem, chlorophyll, vitamin B,,, coenzyme F430, and related tetrapyrrolic pigments. It catalyses the conversion of four equivalents of porphobilinogen (PBG; 2) into (hydroxymethy1)-bilane derivative 3 and ammonia [ 11 [2] (Scheme I ) .HMBS is an enzyme converting PBG into products obeying Michaelis-Menten kinetics (k,,, = 0.1 s-', K,,, = 5-10 ~L M at pH 7.4 for HMBS from Escherichia coli [3]) [4] and displaying chemical reactivity of a polymerase [S]. The signal to stop polymerisation does not require any external factors, but is built into the HMBS molecule itself as soon as four substrate molecules have been processed, the tetrameric product is released. To accomplish the assembly of the bilane, HMBS uses a unique dipyrrin ( = dipyrromethane) cofactor to which the growing chain remains covalently attached [6] [7] (see Scheme 2). The cofactor is in turn covalently bound to the enzyme via the S-atom of a cysteine residue (Cys-242 of the enzyme from E.coli). The HMBS apoenzyme, lacking the cofactor, is capable of assembling its own cofactor from two substrate molecules. Being derived from PBG (2), the cofactor can be isotopically labelled by

The structure of a new red pigment produced during the “bleaching” of Chlorella protorhecoides cells has been elucidated by analytical methods as well as by in vitro isomerization into the new compound of a synthetic sample of a previously characteriLed chlorophyll a catabolite.