Small Angle X-Ray Scattering Study on Bovine Porphobilinogen Synthase (5-Aminolaevulinate Dehydratase)

Pilz, Ingrid; Schwarz, Erika; Vuga, Maximilian; Beyersmann, Detmar

doi:10.1515/bchm3.1988.369.2.1099

Cited by 14 publications

(7 citation statements)

References 6 publications

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“…There are two monomers in the asymmetric unit of human native ALAD, which form a closely associated dimer that, in combination with the crystallographic fourfold axis, forms octamers with approximate dimensions of 108 Â 108 Â 78 Å , in accord with earlier EM studies (Wu et al, 1974;Pilz et al, 1988). The octamer, the assembly of which is shown in Fig.…”

Section: Human Aladsupporting

confidence: 80%

“…The human and bovine ALAD enzymes have been purified in high yield from both blood and liver, where the enzyme is present in high abundance (Gibbs et al, 1985;Jordan & Seehra, 1986). Gel-filtration, sedimentation, electronmicroscopy and solution-scattering studies have shown that the mammalian ALAD enzymes are octameric (Wu et al, 1974;Bevan et al, 1980;Pilz et al, 1988). However, there are reports that the plant enzyme can adopt a hexameric form (see, for example, Kokona et al, 2008), and a hexameric, porphyria-associated mutant form of the human enzyme has been described (Breinig et al, 2003), leading to speculation that interconversion between these oligomeric states provides a means of regulating the activity of ALAD enzymes, as corroborated by further studies (Jaffe & Lawrence, 2012).…”

Section: Figurementioning

confidence: 99%

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Structural studies of substrate and product complexes of 5-aminolaevulinic acid dehydratase from humans,Escherichia coliand the hyperthermophilePyrobaculum calidifontis

Mills-Davies

Butler

Norton

et al. 2017

Acta Cryst Sect D Struct Biol

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A number of X-ray analyses of an enzyme involved in a key early stage of tetrapyrrole biosynthesis are reported. Two structures of human 5-aminolaevulinate dehydratase (ALAD), native and recombinant, have been determined at 2.8 Å resolution, showing that the enzyme adopts an octameric quaternary structure in accord with previously published analyses of the enzyme from a range of other species. However, this is in contrast to the finding that a disease-related F12L mutant of the human enzyme uniquely forms hexamers [Breinig et al. (2003), Nature Struct. Biol. 10, 757-763]. Monomers of all ALADs adopt the TIM-barrel fold; the subunit conformation that assembles into the octamer includes the N-terminal tail of one monomer curled around the (α/β) barrel of a neighbouring monomer. Both crystal forms of the human enzyme possess two monomers per asymmetric unit, termed A and B. In the native enzyme there are a number of distinct structural differences between the A and B monomers, with the latter exhibiting greater disorder in a number of loop regions and in the active site. In contrast, the second monomer of the recombinant enzyme appears to be better defined and the active site of both monomers clearly possesses a zinc ion which is bound by three conserved cysteine residues. In native human ALAD, the A monomer also has a ligand resembling the substrate ALA which is covalently bound by a Schiff base to one of the active-site lysines (Lys252) and is held in place by an ordered active-site loop. In contrast, these features of the active-site structure are disordered or absent in the B subunit of the native human enzyme. The octameric structure of the zinc-dependent ALAD from the hyperthermophile Pyrobaculum calidifontis is also reported at a somewhat lower resolution of 3.5 Å. Finally, the details are presented of a high-resolution structure of the Escherichia coli ALAD enzyme co-crystallized with a noncovalently bound moiety of the product, porphobilinogen (PBG). This structure reveals that the pyrrole side-chain amino group is datively bound to the active-site zinc ion and that the PBG carboxylates interact with the enzyme via hydrogen bonds and salt bridges with invariant residues. A number of hydrogen-bond interactions that were previously observed in the structure of yeast ALAD with a cyclic intermediate resembling the product PBG appear to be weaker in the new structure, suggesting that these interactions are only optimal in the transition state.

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Section: Human Aladsupporting

confidence: 80%

Section: Figurementioning

confidence: 99%

Structural studies of substrate and product complexes of 5-aminolaevulinic acid dehydratase from humans,Escherichia coliand the hyperthermophilePyrobaculum calidifontis

Mills-Davies

Butler

Norton

et al. 2017

Acta Cryst Sect D Struct Biol

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show abstract

“…The lack of discernible spectral alterations when ALA binds suggests that ALA is not directly coordinated to the Mn(II), implying that Mn(I1) is an allosteric activator, not located at the active site. The presence of four active sites per PBGS octamer suggests that the dimer is the fundamental functional unit, and small angle X-ray diffraction data support this model (Pilz et al, 1988). Because both substrate and/or Mg(I1) promote octamer formation, this work was unable to address the activity of smaller species such as the dimer.…”

Section: Discussionmentioning

confidence: 97%

Characterization of the Role of the Stimulatory Magnesium of Escherichia coli Porphobilinogen Synthase

Jaffe¹,

Ali²,

Mitchell³

et al. 1995

Biochemistry

116

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The synthesis of tetrapyrroles is essential to all phyla. Porphobilinogen synthase (PBGS) is a zinc metalloenzyme that catalyzes the formation of porphobilinogen, the monopyrrole precursor of all biological tetrapyrroles. The enzyme from various organisms shows considerable sequence conservation, suggesting a common fold, quaternary structure, and catalytic mechanism. Escherichia coli and plant PBGS are activated by magnesium, a property that is absent from mammalian PBGS. This stimulatory Mg(II) is called Mgc. Mgc is not required for activity and is distinct from the two zinc ions (ZnA and ZnB) common to mammalian and E. coli PBGS (PBGSE.coli). For PBGSE.coli, both the Km for the substrate 5-aminolevulinic acid (ALA) and the Vmax are altered by the presence of Mgc; Mg(II) causes the Km to drop from approximately 3 to 0.30 mM and the maximum specific activity to increase from 23 to 50 mumol h-1 mg-1. Mgc also causes the saturating concentration of the required Zn(II) to decrease from 0.1 mM to 10 microM. Maximal activation by Mg(II) occurs at 0.5 mM; thus, in E. coli the Mgc site is probably saturated under physiological conditions. Mn(II) is a good substitute for Mgc, giving a comparable increase in catalytic activity. Consequently, Mn(II) has been used as an EPR active probe of the Mgc binding site. Mn(II) binds at a stoichiometry of eight ions per enzyme octamer. The X- and Q-band EPR spectra reflect a single type of binding site with rhombic symmetry and are consistent with oxygen and/or nitrogen ligands. The addition of unlabeled or 1-13C-labeled ALA does not significantly affect the Mn(II) EPR spectra.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…Electron microscopy of bovine 5-ALA dehydratase has revealed that the eight subunits are arranged at the comers of a cube with dihedral symmetry (Wu et al, 1974). Low angle X-ray solution scattering indicates that the octamer exists as a quadratic arrangement of four dimeric stacks (Pilz et al, 1988) resembling a slightly squashed cube. These studies have indicated the presence of fourfold symmetry within the octamer (Pilz et al, 1988), the dimensions of which were estimated to be 97 X 97 X 70 A'.…”

mentioning

confidence: 99%

“…The particles appeared to be 85-90 A in all three dimensions. Small angle X-ray scattering studies (Pilz et al, 1988) suggested that the enzyme is less symmetric and can be modeled as a slightly flattened cube with dimensions of around 97 X 97 X 70 A'. From consideration of biochemical data that suggested that the most stable oligomeric component of the octamer is the dimer, it was inferred that these dimers could be arranged around a four-fold axis to generate particles made of four dimers each with its longest dimension roughly parallel with the four-fold axis.…”

mentioning

confidence: 99%

Crystallization of 5‐aminolaevulinic acid dehydratase from Escherichia coli and Saccharomyces cerevisiae and preliminary X‐ray characterization of the crystals

Erskine¹,

Cooper²,

Lambert³

et al. 1997

Protein Science

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Small Angle X-Ray Scattering Study on Bovine Porphobilinogen Synthase (5-Aminolaevulinate Dehydratase)

Cited by 14 publications

References 6 publications

Structural studies of substrate and product complexes of 5-aminolaevulinic acid dehydratase from humans,Escherichia coliand the hyperthermophilePyrobaculum calidifontis

Structural studies of substrate and product complexes of 5-aminolaevulinic acid dehydratase from humans,Escherichia coliand the hyperthermophilePyrobaculum calidifontis

Characterization of the Role of the Stimulatory Magnesium of Escherichia coli Porphobilinogen Synthase

Crystallization of 5‐aminolaevulinic acid dehydratase from Escherichia coli and Saccharomyces cerevisiae and preliminary X‐ray characterization of the crystals

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