Crystallization and preliminary X-ray characterization of the tetrapyrrole-biosynthetic enzyme porphobilinogen deaminase from<i>Bacillus megaterium</i>

Azim, N.; Deery, Evelyne; Warren, Martin J.; Erskine, P.T.; Cooper, J.B.; Wood, Steve; Akhtar, Muhammad

doi:10.1107/s1744309113018526

Cited by 3 publications

(3 citation statements)

References 18 publications

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“…Another interesting feature of B. megaterium PBGD is the pink colouration which is found in the cells expressing the enzyme and in the final affinity-purified enzyme (Azim et al, 2013). Over time the protein solution becomes progressively yellow and the crystals we obtained were indeed of this colour, as is the case for most PBGDs studied to date.…”

Section: Definition Of the Cofactor Redox Statessupporting

confidence: 52%

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Structural evidence for the partially oxidized dipyrromethene and dipyrromethanone forms of the cofactor of porphobilinogen deaminase: structures of the Bacillus megaterium enzyme at near-atomic resolution

Azim

Deery

Warren

et al. 2014

Acta Crystallogr D Biol Crystallogr

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The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses an early step of the tetrapyrrole-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor, which is covalently linked by a thioether bridge to an invariant cysteine residue (Cys241 in the Bacillus megaterium enzyme). The cofactor is extended during the reaction by the sequential addition of the four substrate molecules, which are released as a linear tetrapyrrole product. Expression in Escherichia coli of a His-tagged form of B. megaterium PBGD has permitted the X-ray analysis of the enzyme from this species at high resolution, showing that the cofactor becomes progressively oxidized to the dipyrromethene and dipyrromethanone forms. In previously solved PBGD structures, the oxidized cofactor is in the dipyromethenone form, in which both pyrrole rings are approximately coplanar. In contrast, the oxidized cofactor in the B. megaterium enzyme appears to be in the dipyrromethanone form, in which the C atom at the bridging -position of the outer pyrrole ring is very clearly in a tetrahedral configuration. It is suggested that the pink colour of the freshly purified protein is owing to the presence of the dipyrromethene form of the cofactor which, in the structure reported here, adopts the same conformation as the fully reduced dipyrromethane form.

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Section: Definition Of the Cofactor Redox Statessupporting

confidence: 52%

“…The expression of B. megaterium PBGD in E. coli using a pET-14b construct has been reported recently along with the purification and crystallization of the enzyme (Azim et al, 2013). The affinity-purified enzyme was assayed spectrophotometrically using the method described in Shoolingin- .…”

Section: Methodsmentioning

confidence: 99%

Structural evidence for the partially oxidized dipyrromethene and dipyrromethanone forms of the cofactor of porphobilinogen deaminase: structures of the Bacillus megaterium enzyme at near-atomic resolution

Azim

Deery

Warren

et al. 2014

Acta Crystallogr D Biol Crystallogr

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“…The wild-type B. megaterium PBGD gene in a pET-14b expression construct, as previously reported by Azim et al (2013), was used with the QuikChange mutagenesis kit (Agilent Technologies, Cheshire, England) to introduce the required base changes. The following mutagenic primers were ordered from Yorkshire Bioscience (York Science Park, York, England) with the base changes shown in bold.…”

Section: Mutagenesis Expression and Purificationmentioning

confidence: 99%

Structural studies of domain movement in active-site mutants of porphobilinogen deaminase fromBacillus megaterium

et al. 2017

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The enzyme porphobilinogen deaminase (PBGD) is one of the key enzymes in tetrapyrrole biosynthesis. It catalyses the formation of a linear tetrapyrrole from four molecules of the substrate porphobilinogen (PBG). It has a dipyrromethane cofactor (DPM) in the active site which is covalently linked to a conserved cysteine residue through a thioether bridge. The substrate molecules are linked to the cofactor in a stepwise head-to-tail manner during the reaction, which is catalysed by a conserved aspartate residue: Asp82 in the B. megaterium enzyme. Three mutations have been made affecting Asp82 (D82A, D82E and D82N) and their crystal structures have been determined at resolutions of 2.7, 1.8 and 1.9 Å, respectively. These structures reveal that whilst the D82E mutant possesses the DPM cofactor, in the D82N and D82A mutants the cofactor is likely to be missing, incompletely assembled or disordered. Comparison of the mutant PBGD structures with that of the wild-type enzyme shows that there are significant domain movements and suggests that the enzyme adopts `open' and `closed' conformations, potentially in response to substrate binding.

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The crystal structures of the enzyme hydroxymethylbilane synthase, also known as porphobilinogen deaminase

Helliwell

2021

Acta Cryst Sect F

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The enzyme hydroxymethylbilane synthase (HMBS; EC 4.3.1.8), also known as porphobilinogen deaminase, catalyses the stepwise addition of four molecules of porphobilinogen to form the linear tetrapyrrole 1-hydroxymethylbilane. Thirty years of crystal structures are surveyed in this topical review. These crystal structures aim at the elucidation of the structural basis of the complex reaction mechanism involving the formation of tetrapyrrole from individual porphobilinogen units. The consistency between the various structures is assessed. This includes an evaluation of the precision of each molecular model and what was not modelled. A survey is also made of the crystallization conditions used in the context of the operational pH of the enzyme. The combination of 3D structural techniques, seeking accuracy, has also been a feature of this research effort. Thus, SAXS, NMR and computational molecular dynamics have also been applied. The general framework is also a considerable chemistry research effort to understand the function of the enzyme and its medical pathologies in acute intermittent porphyria (AIP). Mutational studies and their impact on the catalytic reaction provide insight into the basis of AIP and are also invaluable for guiding the understanding of the crystal structure results. Future directions for research on HMBS are described, including the need to determine the protonation states of key amino-acid residues identified as being catalytically important. The question remains – what is the molecular engine for this complex reaction? Thermal fluctuations are the only suggestion thus far.

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Crystallization and preliminary X-ray characterization of the tetrapyrrole-biosynthetic enzyme porphobilinogen deaminase fromBacillus megaterium

Cited by 3 publications

References 18 publications

Structural evidence for the partially oxidized dipyrromethene and dipyrromethanone forms of the cofactor of porphobilinogen deaminase: structures of the Bacillus megaterium enzyme at near-atomic resolution

Structural evidence for the partially oxidized dipyrromethene and dipyrromethanone forms of the cofactor of porphobilinogen deaminase: structures of the Bacillus megaterium enzyme at near-atomic resolution

Structural studies of domain movement in active-site mutants of porphobilinogen deaminase fromBacillus megaterium

The crystal structures of the enzyme hydroxymethylbilane synthase, also known as porphobilinogen deaminase

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