Abstract
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. Expression in Escherichia coli of a His-tagged form of Bacillus megaterium PBGD permitted the crystallization and preliminary X-ray analysis of the enzyme from this species at high resolution.
Highlights
The enzyme porphobilinogen deaminase (PBGD), which is known as 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, preuroporphyrinogen or hydroxymethylbilane (Fig. 1; Jordan, 1991)
The B. megaterium PBGD gene was cloned into the NdeI and BamHI restriction sites of the expression vector pET14b using standard methods and was transformed into Rosetta (DE3) E. coli cells (Novagen)
The cleaved tag and thrombin were removed by passing the previously dialysed protein through a HisTrap HP 1 ml column again followed by a HiTrap Benzamidine FF 1 ml column (GE Healthcare)
Summary
The enzyme porphobilinogen deaminase (PBGD), which is known as 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, preuroporphyrinogen or hydroxymethylbilane (Fig. 1; Jordan, 1991). Isotopic labelling and single-turnover studies showed that the pyrrole-forming ring A (Fig. 1) is the first to bind to the enzyme, followed by rings B, C and D. The enzyme possesses a dipyrromethane cofactor (Fig. 2) which is covalently bound to the enzyme by a thioether linkage involving an invariant cysteine residue (Jordan & Warren, 1987). The cofactor remains covalently attached to the enzyme when the product of the reaction is released. The dipyrromethane cofactor is attached to a loop on domain 3 and is positioned at the mouth of a deep active-site cleft formed between domains 1 and 2. We report the expression and crystallization of PBGD from B. megaterium in a form that diffracts synchrotron radiation to very high resolution
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