Crystallization and preliminary X-ray investigation of Escherichia coli porphobilinogen deaminase

Jordan, Peter M.; Warren, Martin J.; Mgbeje, B I A; Wood, Stephen P.; Cooper, J.B.; Louie, G.V.; Brownlie, Paul D.; Lambert, Richard; Blundell, T.L.

doi:10.1016/0022-2836(92)90590-g

Cited by 19 publications

(15 citation statements)

References 13 publications

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“…Indeed, when enzyme with a 44 % occupancy and a 1 : 1 : 3 distribution of molecules containing six, five and no selenium atoms was crystallised, crystals indistinguishable from those grown with enzyme of more than 99% selenium occupancy were obtained. Both types of crystal produced strong diffraction patterns and were stable in the X-ray beam for at least 40 h, these properties being the same as those reported for crystals of wild-type IIMBS [39]. With space group P21212, we found unit cell dimensions of a = 8.841 nm, b = 7.636 nm, c = 5.065 nm for [SeMetIHMBS with 44% selenium occupancy, and a = 8.847 nm, b = 7.615 nm, c = 5.079 nm for [SeMetIHMBS with more than 99 % selenium occupancy.…”

Section: Crystallisation and X-ray Analysissupporting

confidence: 69%

“…With space group P21212, we found unit cell dimensions of a = 8.841 nm, b = 7.636 nm, c = 5.065 nm for [SeMetIHMBS with 44% selenium occupancy, and a = 8.847 nm, b = 7.615 nm, c = 5.079 nm for [SeMetIHMBS with more than 99 % selenium occupancy. These dimensions agree very well with the cell dimensions a = 8.801 nm, b = 7.586 nm, c = 5.053 nm found for crystals of wild-type HMBS, indicating one molecule of the enzyme in the asymmetric unit [39]. Data were collected from a single crystal of HMBS with more than 99% selenium occupancy using a FAST area detector.…”

Section: Crystallisation and X-ray Analysissupporting

confidence: 64%

“…On storage the crystals became pale yellow, the intensity of the yellow colour increasing with time. The same behaviour was observed with wild-type HMBS crystals [39]. Crystals grown and stored in a system with solid dithiothreitol in the reservoir, as mentioned above, are significantly more stable than crystals grown in exactly the same way but with 200 pl poly-(ethylene glycol) 6000 stock solution added to solid dithiothreitol in the reservoir.…”

Section: Crystallisation and X-ray Analysissupporting

confidence: 62%

“…We tend to take the latter view, for the following reasons. Crystals grown from freshly purified [SeMetIHMBS were colourless, whereas the crystals from which the native structure was solved were light yellow [39]. Active HMBS carrying its cofactor, as shown in Fig.…”

Section: Crystallisation and X-ray Analysismentioning

confidence: 99%

“…Rectangular crystals more suitable for X-ray diffraction analysis were eventually obtained using conditions based on those reported by Jordan et al for the crystallisation of wildtype HMBS [39], with the following modifications. Sitting drops (50 pl) containing 12-14 mg/ml protein, 0.6 mM EDTA and 31 mM dithiothreitol in 0.2M sodium actetate (pH 5.3) were mixed with an equal volume of a 20% (mass/ vol.)…”

Section: Crystallisation and X-ray Analysismentioning

confidence: 99%

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

Hädener

Matzinger

Malashkevich

et al. 1993

European Journal of Biochemistry

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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 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...

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Section: Crystallisation and X-ray Analysissupporting

confidence: 69%

Section: Crystallisation and X-ray Analysissupporting

confidence: 64%

Section: Crystallisation and X-ray Analysissupporting

confidence: 62%

Section: Crystallisation and X-ray Analysismentioning

confidence: 99%

Section: Crystallisation and X-ray Analysismentioning

confidence: 99%

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

Hädener

Matzinger

Malashkevich

et al. 1993

European Journal of Biochemistry

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The three-dimensional structure ofEscherichia coliporphobilinogen deaminase at 1.76-Å resolution

Louie¹,

Brownlie²,

Lambert³

et al. 1996

Proteins

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Porphobilinogen deaminase (PBGD) catalyses the polymerization of four molecules of porphobilinogen to form the 1‐hydroxymethylbilane, preuroporphyrinogen, a key intermediate in the biosynthesis of tetrapyrroles. The three‐dimensional structure of wild‐type PBGD from Escherichia coli has been determined by multiple isomorphous replacement and refined to a crystallographic R‐factor of 0.188 at 1.76 Å resolution. The polypeptide chain of PBGD is folded into three α/β domains. Domains 1 and 2 have a similar overall topology, based on a five‐stranded, mixed β‐sheet. These two domains, which are linked by two hinge segments but otherwise make few direct interactions, form an extensive active site cleft at their interface. Domain 3, an open‐faced, anti‐parallel sheet of three strands, interacts approximately equally with the other two domains. The dipyrromethane cofactor is covalently attached to a cysteine side‐chain borne on a flexible loop of domain 3. The cofactor serves as a primer for the assembly of the tetrapyrrole product and is held within the active site cleft by hydrogen‐bonds and salt‐bridges that are formed between its acetate and propionate side‐groups and the polypeptide chain. The structure of a variant of PBGD, in which the methionines have been replaced with selenomethionines, has also been determined. The cofactor, in the native and functional form of the enzyme, adopts a conformation in which the second pyrrole ring (C2) occupies an internal position in the active site cleft. On oxidation, however, this C2 ring of the cofactor adopts a more external position that may correspond approximately to the site of substrate binding and polypyrrole chain elongation. The side‐chain of Asp84 hydrogen‐bonds the hydrogen atoms of both cofactor pyrrole nitrogens and also potentially the hydrogen atom of the pyrrole nitrogen of the porphobilinogen molecule bound to the proposed substrate binding site. This group has a key catalytic role, possibly in stabilizing the positive charges that develop on the pyrrole nitrogens during the ring‐coupling reactions. Possible mechanisms for the processive elongation of the polypyrrole chain involve: accommodation of the elongating chain within the active site cleft, coupled with shifts in the relative positions of domains 1 and 2 to carry the terminal ring into the appropriate position at the catalytic site; or sequential translocation of the elongating polypyrrole chain, attached to the cofactor on domain 3, through the active site cleft by the progressive movement of domain 3 with respect to domains 1 and 2. Other mechanisms are considered although the amino acid sequence comparisons between PBGDs from all species suggest they share the same three‐dimensional structure and mechanism of activity. © 1996 Wiley‐Liss, Inc.

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Structural Studies on Porphobilinogen Deaminase

Lambert

et al. 2007

Ciba Foundation Symposium 180 ‐ the Biosynthesis of the Tetrapyrrole Pigments

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Crystallization and preliminary X-ray investigation of Escherichia coli porphobilinogen deaminase

Cited by 19 publications

References 13 publications

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

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

The three-dimensional structure ofEscherichia coliporphobilinogen deaminase at 1.76-Å resolution

Structural Studies on Porphobilinogen Deaminase

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