Purification, crystallization and properties of porphobilinogen deaminase from a recombinant strain of <i>Escherichia coli</i> K12

Jordan, Peter M.; Thomas, Steven; Warren, Martin J.

doi:10.1042/bj2540427

Cited by 70 publications

(31 citation statements)

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“…By using poly(ethy1ene glycol) 6000 and a pH value closer to the pZ of the enzyme (allowing a lower protein concentration), as reported by Jordan et al [39] for the crystallisation of wildtype HMBS, the SeMet-containing enzyme could be readily crystallised in another, rectangular crystal form more suitable for X-ray analysis. It must be remembered, however, that the latter conditions sample the pHrange at a point (5.0-5.5) where HMBS exhibits no enzyme activity [41]. Hence, an improvement of the rhombohedral crystal form, which can be obtained at the pH optimum of the enzyme, is highly de-sirable.…”

Section: Resultsmentioning

confidence: 99%

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: Resultsmentioning

confidence: 99%

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 recombinant production of E. coli HmbS allowed the enzyme to be crystallized, and it was the first enzyme of tetrapyrrole biosynthesis to have its structure determined by X-ray crystallography (116,127). This structure revealed that the protein was composed of three domains, with the dipyrromethane cofactor being attached to domain 3 and with the majority of the active site being formed within a cleft between domains 1 and 2 (128,129) ( Fig.…”

Section: Dailey Et Almentioning

confidence: 99%

“…The discovery, isolation, and sequencing of the E. coli hemC gene allowed the enzyme to be overproduced, making the protein much more readily available (116,117). Most HmbSs have a molecular mass of around 35 kDa and are monomeric.…”

Section: Dailey Et Almentioning

confidence: 99%

Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product

Dailey

Gerdes³

et al. 2017

Microbiol Mol Biol Rev

264

335

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SUMMARY The advent of heme during evolution allowed organisms possessing this compound to safely and efficiently carry out a variety of chemical reactions that otherwise were difficult or impossible. While it was long assumed that a single heme biosynthetic pathway existed in nature, over the past decade, it has become clear that there are three distinct pathways among prokaryotes, although all three pathways utilize a common initial core of three enzymes to produce the intermediate uroporphyrinogen III. The most ancient pathway and the only one found in the Archaea converts siroheme to protoheme via an oxygen-independent four-enzyme-step process. Bacteria utilize the initial core pathway but then add one additional common step to produce coproporphyrinogen III. Following this step, Gram-positive organisms oxidize coproporphyrinogen III to coproporphyrin III, insert iron to make coproheme, and finally decarboxylate coproheme to protoheme, whereas Gram-negative bacteria first decarboxylate coproporphyrinogen III to protoporphyrinogen IX and then oxidize this to protoporphyrin IX prior to metal insertion to make protoheme. In order to adapt to oxygen-deficient conditions, two steps in the bacterial pathways have multiple forms to accommodate oxidative reactions in an anaerobic environment. The regulation of these pathways reflects the diversity of bacterial metabolism. This diversity, along with the late recognition that three pathways exist, has significantly slowed advances in this field such that no single organism's heme synthesis pathway regulation is currently completely characterized.

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“…to uroporphyrinogen III, the precursor for haem, chlorophyll and vitamin B-12 (Battersby et al, 1980). HMBS has previously been purified from Escherichia coli Jordan et al, 1988) and the corresponding gene hemC has been sequenced (Thomas & Jordan, 1986;Alefounder et al, 1988).…”

Section: Introductionmentioning

confidence: 99%

Investigation of putative active-site lysine residues in hydroxymethylbilane synthase. Preparation and characterization of mutants in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 have been replaced by glutamine

Hädener

Alefounder

Hart

et al. 1990

Biochemical Journal

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A new construct carrying the hemC gene was transformed into Escherichia coli, resulting in approx. 1000-fold overexpression 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 kaCpp IKapp . 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.

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Purification, crystallization and properties of porphobilinogen deaminase from a recombinant strain of Escherichia coli K12

Cited by 70 publications

References 30 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

Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product

Investigation of putative active-site lysine residues in hydroxymethylbilane synthase. Preparation and characterization of mutants in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 have been replaced by glutamine

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