Porphyrin biosynthesis in rhodopseudomonas palustris—V. Purification of porphyrinogen decarboxylase and some unusual properties

Abstract

A~tract--Uroporphyrinogen decarboxylase (EC 4.1.1.37) has been purified 16-fold from Rp. palustr• to a specific activity of 210 nmol of total decarboxylated porphyrinogens III formed/hr per mg of protein and about 50% yield. The Rp. palustris enzyme exhibits some unusual properties as compared with URO-D from other sources.2. The purified enzyme is a monomer with a molecular weight of ~46,000, an isoelectric point of 4.6 and an optimum pH of 6.9 and 6.8 with urogen III and I substrate. Neither GSH nor EDTA seem to be necessary for activity, and the decarboxylation rate and the distribution of the reaction products was not affected either by the presence or absence of oxygen.3. The Rp. palustris enzyme is a thermo-stable protein, heating at 60°C for 15 min enhanced several times activity. This is the first time that heat treatment is included as one of the steps to purify URO-D. 4. Thermal activation followed an identical profile using either substrate. The ratios of specific activity for the type III and I isomer of urogen remained constant throughout the purification. These findings are indicating that a single enzyme catalyzes the four decarboxylations occurring from urogen to coprogen.5. Kinetic data employing urogen III and I as substrate showed that the pattern of accumulated intermediates was rather different depending on whether type III or I isomer was used.6. While decarboxylation of urogen III responds to the usual scheme:' octagen III "' , heptagen III ": , coprogen III, where v t ~> v2 and decarboxylation of heptagen III is the rate-controlling step. 7. Decarboxylation of urogen I revealed a completely different and characteristic picture fitting the scheme: octagen I '~ , pentagen I '~ , coprogen I, where again v'~ ~> v~ and the removal of the final carboxyl group from pentagen I becomes the rate-limiting step.

Studies on uroporphyrinogen decarboxylase fromChlorella kessleri(Trebouxiophyceae, Chlorophyta)

“…This is the first time that UroD activity has been studied in a green algae (Chlorella). The chromatographic profile of the reaction products is similar to that of UroD from other sources, such as rat liver (Ríos de Molina et al 1987), bacteria (Koopmann et al 1986), and Euglena gracilis (Juknat et al 1989). Moreover, this profile shows the prevalence of 7 COOH over 6 and 5 COOH intermediates, suggesting that 7 COOH porphyrinogen decarboxylation is the rate-limiting step in C. kessleri UroD reaction, as reported for rat and bovine liver (Straka and Kushner 1983;Ríos de Molina et al 1987), avian erythrocytes (García et al 1973), and bacteria (Koopmann et al 1986)…”

“…During the first stage, the rapid decarboxylation of Urogen III (8 COOH) yields a 7 COOH compound. During the second, the other 3 COOH groups are sequentially eliminated yielding coproporphyrinogen III (Coprogen III, 4 COOH) through the formation of 6 and 5 COOH porphyrinogen intermediates (Jackson et al 1976; Koopmann et al 1986).…”

Purification and properties of the uroporphyrinogen decarboxylase from Rhodobacter sphaeroides

“…The effects of heat upon activity are similar to those made on the uroporphyrinogen decarboxylases from human erythrocytes (Elder et al, 1983;de Verneuil et al, 1983a). In contrast, the uroporphyrinogen decarboxylase partially purified from R. palustrus (Koopmann et al, 1986) was reported to be heat stable and this property was exploited during its partial purification.…”

“…Previously published methods describe only the partial purification of the enzyme from R. sphaeroides (Hoare and Heath, 1959) and R. palustrus (Koopmann et al, 1986). The purification procedure described in this paper gives homogeneous uroporphyrinogen decarboxylase from R. sphaeroides, with a 600-fold purification overall (Table 1).…”

“…The reaction proceeds through intermediate porphyrinogens with seven, six and five carboxyl groups (Scheme 1). Uroporphyrinogen decarboxylase has been purified to homogeneity from human erythrocytes (de Verneuil et al, 1983a;Elder et al, 1983), avian erythrocytes (Kawanishi et al, 1983), bovine liver (Straka and Kushner, 1983) and yeast (Felix and Brouillet, 1990), and has been partially purified from tobacco leaves (Chen and Miller, 1974) and the bacterium Rhodobacter palustrus (Koopmann et al, 1986).…”