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

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.     

Spectroscopic evidence for a porphobilinogen deaminase-tetrapyrrole complex that is an intermediate in the biosynthesis of uroporphyrinogen III

Incubation of porphobilinogen (PBG) with PBG deaminase from Rhodopseudomonas sphaeroides in carbonate buffer (pH 9.2) to total PBG consumption resulted in low yields of uroporphyrinogen I (uro'gen I). In the reaction mixture a pyrrylmethane accumulated, which at longer incubation periods was transformed into uro'gen I. The accumulated pyrrylmethane gave an Ehrlich reaction which was different from that of a 2-(aminomethyl)dipyrrylmethane or 2-(aminomethyl)tripyrrane. It resembled that of a bilane (tetrapyrrylmethane) but was different from that of a 2-(hydroxymethyl)bilane. The 13C NMR spectra of incubations carried out with [11-13C]PBG indicated that the pyrrylmethane was a tetrapyrrole with methylene resonances at 22.35-22.50 ppm. It was loosely bound to the deaminase, and when separated from the enzyme by gel filtration or gel electrophoresis, it immediately cyclized to uro'gen I. No enzyme-bound methylene could be detected by its chemical shift, suggesting that its line width must be very broad. When uro'gen III-cosynthase was added to the deaminase-tetrapyrrole complex, uro'gen III was formed at the expense of the latter in about 75% yield. The tetrapyrrole could only be partially displaced from the enzyme by ammonium ions, although a small amount of 2-(aminomethyl)bilane was always formed together with the tetrapyrrole intermediate. A protonated uro'gen I structure for this intermediate was ruled out by incubations using [2,11-13C]PBG. Uro'gen III formation from 2-(hydroxymethyl)bilane (HMB) and from the deaminase-tetrapyrrole intermediate was compared by using deaminase-cosynthase and cosynthase from several sources. It was found that while the HMB inhibited uro'gen III formation at higher concentrations and longer incubation times, uro'gen III formation from the complex did not decrease with time.     

Porphyrin biosynthesis from porphobilinogen by duck blood hemolysate

Summary The formation of porphyrins from porphobilinogen by a duck blood hemolysate was examined. The system was found to form mainly protoporphyrin IX and hemin, and accumulated lesser amounts of uroporphyrins, heptacarboxylic porphyrin, and coproporphyrins. By storage at –20° the accumulation of uroporphyrins and heptacarboxylic porphyrin was increased. Both porphyrins were mainly the type III isomers. By addition of dithiothreitol the porphyrin pattern reversed to the original one formed by the fresh hemolysate. Addition of a number of amines also inhibited the decarboxylating system without affecting the original isomer distribution among the porphyrins. Addition of Fe²⁺ (3mm) did not affect the porphyrin pattern or the isomer distribution. Addition of Pb²⁺ (2.5mm) partially inhibited the decarboxylating system, whereas at higher concentrations (4mm) it increased the decarboxylation rate of the heptacarboxylic porphyrin. The obtained results are discussed in relation to porphyrin accumulation in porphyria cutanea tarda and in acquired hepatic porphyrias.Dedicated to professorLuis F. Leloir on the occasion of his 70th birthday.     

Mechanism of action of porphobilinogen deaminase. The participation of stable enzyme substrate covalent intermediates between porphobilinogen and the porphobilinogen deaminase from Rhodopseudomonas spheroides.

Highly stable labelled complexes are formed between porphobilinogen deaminase and stoicheiometric amounts of [14C]porphobilinogen. On completion of the catalytic cycle by the addition of excess of substrate, the complexes yield labelled product and display all the properties expected from covalently bound enzyme intermediates involved in the deaminase catalytic sequence.     

Control of tetrapyrrole biosynthesis by alternate quaternary forms of porphobilinogen synthase

Porphobilinogen synthase (PBGS) catalyzes the first common step in the biosynthesis of tetrapyrroles (such as heme and chlorophyll). Although the predominant oligomeric form of this enzyme, as inferred from many crystal structures, is that of a homo-octamer, a rare human PBGS allele, F12L, reveals the presence of a hexameric form. Rearrangement of an N-terminal arm is responsible for this oligomeric switch, which results in profound changes in kinetic behavior. The structural transition between octamer and hexamer must proceed through an unparalleled equilibrium containing two different dimer structures. The allosteric magnesium, present in most PBGS, has a binding site in the octamer but not in the hexamer. The unprecedented structural rearrangement reported here relates to the allosteric regulation of PBGS and suggests that alternative PBGS oligomers may function in a magnesium-dependent regulation of tetrapyrrole biosynthesis in plants and some bacteria.     

Involvement of free and enzyme-bound intermediates in the reaction mechanism catalyzed by the bovine liver immobilized porphobilinogen deaminase. Proof that they are substrates for cosynthetase in uroporphyrinogen III biosynthesis

The detection and accumulation of tetrapyrrole intermediates synthesized by the action of bovine liver porphobilinogen deaminase immobilized to Sepharose 4B is reported. Employing Sepharose-deaminase preparations, two phases in uroporphyrinogen I synthesis as a function of time were observed, suggesting the accumulation of free and enzyme-bound intermediates, the concentration and distribution of which were time dependent. The deaminase-bound intermediate behaves as a substrate in uroporphyrinogen I synthesis whereas the free intermediates produce enzyme inhibition. The tetrapyrrole intermediate bound to the Sepharose-enzyme is removed from the protein by the binding of porphobilinogen. Free as well as enzyme-bound intermediates are shown to be substrates for cosynthetase with formation of 80% uroporphyrinogen III.     

Rat hepatic uroporphyrinogen III co-synthase. Purification and evidence for a bound folate coenzyme participating in the biosynthesis of uroporphyrinogen III.

Rat hepatic uroporphyrinogen III co-synthase was isolated and purified 73-fold with a 13% yield by (NH4)2SO4 fractionation and sequential chromatography on DEAE-Sephacel, Sephadex G-100 (superfine grade) and folate-AH-Sepharose 4B. The purified co-synthase has an Mr of approx. 42 000, and is resolved into two bands, each possessing co-synthase activity, by polyacrylamide-gel electrophoresis. A factor was dissociated from the purified co-synthase. Results of both microbiological and competitive protein-binding assays suggest that it is a pteroylpolyglutamate. The isolated pteroylpolyglutamate factor was co-eluted with authentic N5-methyltetrahydropteroylheptaglutamate on DEAE-Sephacel. Uroporphyrinogen III is formed by cosynthase-free preparations of uroporphyrinogen I synthase in the presence of tetrahydropteroylglutamate. Tetrahydropeteroylheptaglutamate is also able to direct the formation of equivalent amounts of uroporphyrinogen III at a concentration approximately one-hundredth that of tetrahydropteroylmonoglutamate. These results suggest that a reduced pteroylpolyglutamate factor is associated with rat hepatic uroporphyrinogen III co-synthase, and that this may function as a coenzyme for the biosynthesis of uroporphyrinogen III.     

Immobilized uroporphyrinogen I synthetase from Rhodopseudomonas palustris

Rhodopseudomonas palustris uroporphyrinogen I synthetase (URO-S) has been chemically attached to Sepharose 4B and some of its properties have been studied. When 7-8 mg protein/ml activated Sepharose was used, immobilized URO-S retained 45% of the activity of the original soluble preparation, with a coupling yield of 66% after a period of 15 h. Optimal incubation conditions for the activity of gel-enzyme were determined. Unlike the soluble enzyme, the Sepharose-bound URO-S showed a biphasic substrate saturation curve, indicating that a protein conformational change had occurred during the process of immobilization. Immobilized URO-S stored at 4 degrees C for 35 days retained 90% of activity and when repeatedly used, up to 5 times, retained 48% of the original activity. Attachment of URO-S to Sepharose led to an enhanced thermal stability.     

Characterization of porphobilinogen deaminase from rat liver

Porphobilinogen deaminase (porphobilinogen ammonia-lyase, EC 4.3.1.8) was isolated from rat liver. The final preparation was homogeneous according to polyacrylamide gel electrophoresis and immunodiffusion criteria. Electrophoresis of the native enzyme revealed a single band of activity which was distributed into three bands after incubation with porphobilinogen. When electrophoresed under denaturing condition it displayed a single polypeptide band with a molecular weight of 42,000 confirmed by exclusion chromatography and by sucrose density gradient centrifugation. The enzyme showed a pH optimum of 7.5 both in 0.1 M sodium phosphate and 0.05 M Tris-HCl buffer, when assayed at 37 degrees C. An isoelectric point of 4.9 for the native purified protein was found. Hepatic porphobilinogen deaminase was remarkably heat-stable showing maximum activity at 55-60 degrees C with one break in the Arrhenius plot. The kinetic behaviour of the purified enzyme followed the typical Michaelis-Menten kinetics with values of Km = 17 microM and Vmax = 29.4 units power mg in 0.1 M phosphate buffer at 37 degrees C. The amino acid composition was determined, showing that the enzyme had a low content of sulphur-containing amino acids and a considerable number of acidic residues per mol of polypeptide chain. Reagents known to interact with sulphydryl groups have small effect on rat liver enzyme activity.     

Crystal structure of uroporphyrinogen decarboxylase from Bacillus subtilis

Uroporphyrinogen decarboxylase (UROD) is a branch point enzyme in the biosynthesis of the tetrapyrroles. It catalyzes the decarboxylation of four acetate groups of uroporphyrinogen III to yield coproporphyrinogen III, leading to heme and chlorophyll biosynthesis. UROD is a special type of nonoxidative decarboxylase, since no cofactor is essential for catalysis. In this work, the first crystal structure of a bacterial UROD, Bacillus subtilis UROD (UROD(Bs)), has been determined at a 2.3 A resolution. The biological unit of UROD(Bs) was determined by dynamic light scattering measurements to be a homodimer in solution. There are four molecules in the crystallographic asymmetric unit, corresponding to two homodimers. Structural comparison of UROD(Bs) with eukaryotic URODs reveals a variation of two loops, which possibly affect the binding of substrates and release of products. Structural comparison with the human UROD-coproporphyrinogen III complex discloses a similar active cleft, with five invariant polar residues (Arg29, Arg33, Asp78, Tyr154, and His322) and three invariant hydrophobic residues (Ile79, Phe144, and Phe207), in UROD(Bs). Among them, Asp78 may interact with the pyrrole NH groups of the substrate, and Arg29 is a candidate for positioning the acetate groups of the substrate. Both residues may also play catalytic roles.