Polypyrroles formed from porphobilinogen and amines by uroporphyrinogen synthetase of Rhodopseudomonas spheroides

Abstract

1. Uroporphyrinogen I synthetase of Rhodopseudomonas spheroides was purified more than 200-fold from the soluble protein of broken bacterial cells. The enzyme had molecular weight 36000, an isoelectric point of 4.46 and migrated as a single active protein band on disc-gel electrophoresis at pH7.5 and 8.9. 2. The enzyme consumed porphobilinogen and formed uroporphyrinogen at pH8.2 without the accumulation of intermediates. In the presence of hydroxylamine, ammonia or methoxyamine the production of porphyrinogen was inhibited and the enzyme formed open-chain polypyrroles instead. 3. These polypyrroles behaved like uroporphyrinogen on Sephadex G-25; they were colourless and had unsubstituted alpha-pyrrolic positions. The inhibitory amines were incorporated into the molecules. 4. The polypyrroles formed porphyrins non-enzymically and the cyclization reaction was accompanied by the release of the inhibitory amine. Exchange of the amino function of the original porphobilinogen in the polypyrrole was complete with hydroxylamine and almost complete with methoxyamine, both ammonia and methoxyamine being present in the polypyrrolic material. 5. The behaviour, properties and composition of the radioactive hydroxylamine derivative were consistent with a tetrapyrrolic structure, probably a pyrrylmethane, that was not cyclized, rather than with di-, tri- or penta-pyrrolic structures. No monopyrrolic or dipyrrolic Ehrlich-positive material was released on cyclization. The ammonia and methoxyamine derivatives had properties similar to the hydroxylamine derivative. 6. Another modified pyrrole was detected only in experiments with hydroxylamine. It differed from both porphobilinogen and known dipyrroles and appeared to be a monopyrrole. 7. The participation of positively charged reaction centres in the enzymic mechanism, particularly in the cyclization step, is discussed.

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

“…E, ES, ES2, ES3, and ES4. Such complexes are susceptible to treatment with hydroxylamine (Davis & Neuberger, 1973) and under these conditions the bound intermediate is released regenerating the free enzyme. The purified E. coli deaminase was therefore treated with 0.2 M-hydroxylamine and re-analysed by both non-denaturing gel electrophoresis and f.p.l.c.…”

Characterization of the Porphobilinogen Deaminase Deficiency in Acute Intermittent Porphyria

“…Increasing attention has focused on the characterization of PBG-deaminase from various sources (13)(14)(15)(16)(17)(18)(19) and the mechanism by which this monome, zc enzyme (-37,000 mol wt) converts the monopyrrole substrate, PBG, to the final tetrapyrrole product, uroporphyrinogen L (20)(21)(22)(23)(24)(25)(26)(27). Recently, we reported the first purification of homogeneous PBG-deaminase from human erythrocytes (27).…”

Purification and kinetic studies on a porphobilinogen deaminase from the unicellular green alga Scenedesmus obliquus

“…The PBG-D has been purified to homogeneity from Escherichia coli (Hart et al 1986;Jordan et al 1988), Euglena gracilis (Battersby et al 1983), Chlorella reyularis (Shioi et al 1980), Rhodopseudomonas spheroides (Davies and Neuberger 1973;Jordan and Shemin 1973), rat spleen (Williams 1984), human erythrocytes (Anderson and Desnick 1980;Smythie and Williams 1988), spinach (Higuchi and Bogorad 1975) and pea chloroplasts (Spano and Timko 1991). Additionally, PBG-D isolated from E. coli has been shown to contain a dipyrromethane cofactor, onto which the extending tetrapyrrole chain is sequentially assembled (Hart et al 1987;Jordan and Warren 1987;Miller et al 1988).…”