先天性红细胞生成性卟啉症患者酶活性的研究

先天性红细胞生成性卟啉症（ｃｏｎｇｅｎｉｔａｌｅｒｙ－ｔｈｒｏｐｏｉｅｔｉｃｐｏｒｐｈｙｒｉａ,ＣＥＰ）是Ｇｕｎｔｈｅｒ于１９１１年首先提出并加以描述,有时亦称Ｇｕｎｔｈｅｒ病．该病是因遗传性缺陷所致卟啉代谢中有关酶的异常造成的卟啉代谢紊乱而发生的一...     

In Vitro Optimization of Enzymes Involved in Precorrin-2 Synthesis Using Response Surface Methodology

In order to maximize the production of biologically-derived chemicals, kinetic analyses are first necessary for predicting the role of enzyme components and coordinating enzymes in the same reaction system. Precorrin-2 is a key precursor of cobalamin and siroheme synthesis. In this study, we sought to optimize the concentrations of several molecules involved in precorrin-2 synthesis in vitro: porphobilinogen synthase (PBGS), porphobilinogen deaminase (PBGD), uroporphyrinogen III synthase (UROS), and S-adenosyl-l-methionine-dependent urogen III methyltransferase (SUMT). Response surface methodology was applied to develop a kinetic model designed to maximize precorrin-2 productivity. The optimal molar ratios of PBGS, PBGD, UROS, and SUMT were found to be approximately 1:7:7:34, respectively. Maximum precorrin-2 production was achieved at 0.1966 ± 0.0028 μM/min, agreeing with the kinetic model’s predicted value of 0.1950 μM/min. The optimal concentrations of the cofactor S-adenosyl-L-methionine (SAM) and substrate 5-aminolevulinic acid (ALA) were also determined to be 200 μM and 5 mM, respectively, in a tandem-enzyme assay. By optimizing the relative concentrations of these enzymes, we were able to minimize the effects of substrate inhibition and feedback inhibition by S-adenosylhomocysteine on SUMT and thereby increase the production of precorrin-2 by approximately five-fold. These results demonstrate the effectiveness of kinetic modeling via response surface methodology for maximizing the production of biologically-derived chemicals.     

Identification and characterization of the Arabidopsis gene encoding the tetrapyrrole biosynthesis enzyme uroporphyrinogen III synthase

UROS (uroporphyrinogen III synthase; EC 4.2.1.75) is the enzyme responsible for the formation of uroporphyrinogen III, the precursor of all cellular tetrapyrroles including haem, chlorophyll and bilins. Although UROS genes have been cloned from many organisms, the level of sequence conservation between them is low, making sequence similarity searches difficult. As an alternative approach to identify the UROS gene from plants, we used functional complementation, since this does not require conservation of primary sequence. A mutant of Saccharomyces cerevisiae was constructed in which the HEM4 gene encoding UROS was deleted. This mutant was transformed with an Arabidopsis thaliana cDNA library in a yeast expression vector and two colonies were obtained that could grow in the absence of haem. The rescuing plasmids encoded an ORF (open reading frame) of 321 amino acids which, when subcloned into an Escherichia coli expression vector, was able to complement an E. coli hemD mutant defective in UROS. Final proof that the ORF encoded UROS came from the fact that the recombinant protein expressed with an N-terminal histidine-tag was found to have UROS activity. Comparison of the sequence of AtUROS (A. thaliana UROS) with the human enzyme found that the seven invariant residues previously identified were conserved, including three shown to be important for enzyme activity. Furthermore, a structure-based homology search of the protein database with AtUROS identified the human crystal structure. AtUROS has an N-terminal extension compared with orthologues from other organisms, suggesting that this might act as a targeting sequence. The precursor protein of 34 kDa translated in vitro was imported into isolated chloroplasts and processed to the mature size of 29 kDa. Confocal microscopy of plant cells transiently expressing a fusion protein of AtUROS with GFP (green fluorescent protein) confirmed that AtUROS was targeted exclusively to chloroplasts in vivo.     

On uroporphyrinogen formation: Studies with 1-aminomethyl-3, 8, 13, 18-tetra(2-carboxyethyl)-2, 7, 12, 17-tetracarboxymethylbilinogen (author's transl)]

The preparation of the aminomethyl-bilinogen which results from formal "head to tail" condensation of porphobilinogen is described. The chemical cyclocondensation of this compound at pH 7.4 yields uroporphyrinogen I. Enzymatic studies with enzyme preparations from Propionibacterium shermanii, which synthesize uroporphyrinogens from porphobilinogen, show that the rate of cyclisation is increased by these enzymes and indicate that the bilinogen also might be used for uroporphyrinogen III formation. This is also suggested by studies on the formation of cobyrinic acid from [4-14C]5-aminolevulinate via uroporphyrinogen III in the presence of the aminomethylbilinogen by cell-free extracts from Clostridium tetanomorphum.     

The common origins of the pigments of life—early steps of chlorophyll biosynthesis

The complex pathway of tetrapyrrole biosynthesis can be dissected into five sections: the pathways that produce 5-aminolevulinate (the C-4 and the C-5 pathways), the steps that transform ALA to uroporphyrinogen III, which are ubiquitous in the biosynthesis of all tetrapyrroles, and the three branches producing specialized end products. These end products include corrins and siroheme, chlorophylls and hemes and linear tetrapyrroles. These branches have been subjects of recent reviews. This review concentrates on the early steps leading up to uroporphyrinogen III formation which have been investigated intensively in recent years in animals, in plants, and in a wide range of bacteria.Abbreviations ALA 5-aminolevulinic acid - ALAS 5-aminolevulinic acid synthase - GR glutamyl-tRNA reductase - GSA glutamate-1-semialdehyde - GSAT glutamate-1-semialdehyde aminotransferase - HMB hydroxymethylbilane - PBG porphobilinogen - PBGD porphobilinogen deaminase - PBGS porphobilinogen synthase - URO uroporphyrin - URO'gen uroporphyrinogen - US uroporphyrinogen III synthase     

Production of uroporphyrinogen III, which is the common precursor of all tetrapyrrole cofactors, from 5-aminolevulinic acid by Escherichia coli expressing thermostable enzymes

Uroporphyrinogen III (urogen III) was produced from 5-aminolevulinic acid (ALA), which is a common precursor of all metabolic tetrapyrroles, using thermostable ALA dehydratase (ALAD), porphobilinogen deaminase (PBGD), and urogen III synthase (UROS) of Thermus thermophilus HB8. The UROS-coding gene (hemD ₂ ) of T. thermophilus HB8 was identified by examining the gene product for its ability to produce urogen III in a coupled reaction with ALAD and PBGD. The genes encoding ALAD, PBGD, and UROS were separately expressed in Escherichia coli BL21 (DE3). To inactivate indigenous mesophilic enzymes, the E. coli transformants were heated at 70 °C for 10 min. The bioconversion of ALA to urogen III was performed using a mixture of heat-treated E. coli transformants expressing ALAD, PBGD, and UROS at a cell ratio of 1:1:1. When the total cell concentration was 7.5 g/l, the mixture of heat-treated E. coli transformants could convert about 88 % 10 mM ALA to urogen III at 60 °C after 4 h. Since eight ALA molecules are required for the synthesis of one porphyrin molecule, approximately 1.1 mM (990 mg/l) urogen III was produced from 10 mM ALA. The present technology has great potential to supply urogen III for the biocatalytic production of vitamin B₁₂.     

Purification and characterization of bovine hepatic uroporphyrinogen decarboxylase

Uroporphyrinogen decarboxylase (EC 4.1.1.37) has been purified to homogeneity from bovine liver by using isoelectric and salt precipitations, followed by chromatography on DEAE-cellulose, phenyl-Sepharose, hydroxylapatite, and Sephacryl S-200. The purified enzyme is a monomer with an Mr approximately 57 000 and an isoelectric point at pH 4.6. Enzyme activity is optimal in buffers having an ionic strength of approximately 0.1 M and a pH of 6.8. The purified enzyme has a specific activity (expressed as the disappearance of uroporphyrinogen I) of 936 nmol X h-1 X (mg of protein)-1. The purified enzyme catalyzes all four decarboxylation reactions in the conversion of uroporphyrinogen I or III to the corresponding coproporphyrinogen. The rate-limiting step in the physiologically significant conversion of uroporphyrinogen III to coproporphyrinogen III is the decarboxylation of heptacarboxylate III. Kinetic data suggest that the enzyme has at least two noninteracting active sites. At least one sulfhydryl group is required for catalytic activity. The enzyme is inhibited by sulfhydryl-specific reagents and by divalent metal ions including Fe2+, Co2+, Cu2+, Zn2+, and Pb2+. The pattern of accumulation of intermediate (hepta-, hexa-, and pentacarboxylate porphyrinogens) and final (coproporphyrinogen) decarboxylation products is affected by the ratio of substrate (uroporphyrinogen I or III) concentration to enzyme concentration. Under physiologic conditions where the uroporphyrinogen to enzyme ratio is low, the substrate is nearly quantitatively decarboxylated, and the major product is coproporphyrinogen. If the ratio of uroporphyrinogen to enzyme is high, intermediates accumulate, and heptacarboxylate porphyrinogen becomes the major decarboxylation product.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rat hepatic uroporphyrinogen III cosynthase: Purification,properties, and inhibition by metal ions

Rat hepatic uroporphyrinogen III cosynthase has been isolated and purified 50-fold with a 36% yield by ammonium sulfate fractionation and sequential chromatography on DEAE-Sephacel and Sephadex G-100SF. Inhibition of uroporphyrinogen III formation with increasing porphobilinogen concentration was observed. Cosynthase was shown to be thermolabile, and a time-dependent loss of enzyme activity during reaction with uroporphyrinogen I synthase and porphobilinogen was observed. The pH optimum for the complete system (synthase and cosynthase) was pH 7.8 in 50 mm Tris-HCl or 50 mm sodium phosphate buffer. Various metals (KCl, NaCl, MgCl₂, CaCl₂) increased formation of Uroporphyrinogen III. Heavy metals including ZnCl₂, CdCl₂, and CuCl₂ were shown to selectively inhibit cosynthase activity, whereas other metals (HgCl₂, PbCl₂) were less selective and inhibited both synthase and cosynthase at similar concentrations.     

Purification and characterization of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Pseudomonas denitrificans.

S-Adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT), the enzyme of the cobalamin biosynthetic pathway which catalyzes C methylation of uroporphyrinogen III, was purified about 150-fold to homogeneity from extracts of a recombinant strain of Pseudomonas denitrificans derived from a cobalamin-overproducing strain by ammonium sulfate fractionation, anion-exchange chromatography, and hydroxyapatite chromatography. The purified protein has an isoelectric point of 6.4 and molecular weights of 56,500 as estimated by gel filtration and 30,000 as estimated by gel electrophoresis under denaturing conditions, suggesting that the active enzyme is a homodimer. It does not contain a chromophoric prosthetic group and does not seem to require metal ions or cofactors for activity. SUMT catalyzes the two successive C-2 and C-7 methylation reactions involved in the conversion of uroporphyrinogen III to precorrin-2 via the intermediate formation of precorrin-1. In vitro studies suggest that the intermediate monomethylated product (precorrin-1) is released from the protein and then added back to the enzyme for the second C-methylation reaction. The pH optimum was 7.7, the Km values for S-adenosyl-L-methionine and uroporphyrinogen III were 6.3 and 1.0 microM, respectively, and the turnover number was 38 h-1. The enzyme activity was shown to be completely insensitive to feedback inhibition by cobalamin and corrinoid intermediates tested at physiological concentration. At uroporphyrinogen III concentrations above 2 microM, SUMT exhibited a substrate inhibition phenomenon. It is suggested that this property might play a regulatory role in cobalamin biosynthesis in the cobalamin-overproducing strain studied.     

Genetic and biochemical characterization of mutants of Saccharomyces cerevisiae blocked in six different steps of heme biosynthesis

Summary Heme-deficient mutants of Saccharomyces cerevisiae have been isolated from two isogenic strains with the use of an enrichment method based on photodynamic properties of Zn-protoporphyrin. They defined seven non-overlapping complementation groups. A mutant representative of each group was further analysed. Genetic analysis showed that each mutant carried a single nuclear recessive mutation. Biochemical studies showed that the observed accumulation and/or excretion of the different heme synthesis precursors by the mutant cells correlated well with the enzymatic deficiencies measured in acellular extracts. Six of the seven mutants were blocked in a different enzyme activity: 5-aminolevulinate synthase, porphobilinogen synthase, uroporphyrinogen I synthase, uroporphyrinogen decarboxylase, coproporphyrinogen III oxidase and ferrochelatase. The other mutant had the same phenotype as the mutant deficient in ferrochelatase activity. However, it possessed a normal ferrochelatase activity when measured in vitro, so this mutant was assumed to be deficient in protoporphyrinogen oxidase activity or in the transport and/or reduction of iron.The absence of PBG synthesis led to a total lack of uroporphyrinogen I synthase activity. The absence of heme, the end product, led to an important increase of coproporphyrinogen III oxidase activity, while the activity of 5-aminolevulinate synthase, the first enzyme of the pathway, was not changed. These results are discussed in terms of possible modes of regulation of heme synthesis pathway in yeast.     

Subcellular localization of two porphyrin-synthesis enzymes in Pisum sativum (pea) and Arum (cuckoo-pint) species.

The subcellular location of the two porphyrin-synthesis enzymes 5-aminolaevulinate dehydratase (ALAD) and porphobilinogen deaminase (PBGD) was investigated in Pisum sativum (pea) leaves and spadices of Arum (cuckoo-pint). Throughout the tissue-fractionation procedures the distribution of the two enzymes paralleled that of the plastid marker enzyme (ADP-glucose pyrophosphorylase), even in Arum, a tissue where the synthesis of non-plastid haem is predominant. The distribution of cytosolic marker enzyme (lactate dehydrogenase) was significantly different from that of ALAD and PBGD and, although purified mitochondria from both species had some residual activity, this was always less than contaminating plastid marker enzyme. The results suggest that ALAD and PBGD are exclusively plastid enzymes. The significance of this for the role of plastids in cellular porphyrin synthesis is discussed.     

Structural basis of pyrrole polymerization in human porphobilinogen deaminase

Human porphobilinogen deaminase (PBGD), the third enzyme in the heme pathway, catalyzes four times a single reaction to convert porphobilinogen into hydroxymethylbilane. Remarkably, PBGD employs a single active site during the process, with a distinct yet chemically equivalent bond formed each time. The four intermediate complexes of the enzyme have been biochemically validated and they can be isolated but they have never been structurally characterized other than the apo- and holo-enzyme bound to the cofactor. We present crystal structures for two human PBGD intermediates: PBGD loaded with the cofactor and with the reaction intermediate containing two additional substrate pyrrole rings. These results, combined with SAXS and NMR experiments, allow us to propose a mechanism for the reaction progression that requires less structural rearrangements than previously suggested: the enzyme slides a flexible loop over the growing-product active site cavity. The structures and the mechanism proposed for this essential reaction explain how a set of missense mutations result in acute intermittent porphyria.     

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.     

Biosynthesis of uroporphyrinogens from porphobilinogen: mechanism and the nature of the process.

The enzymic self-polymerization of prophobilinogen gives rise to the cyclic tetrapyrroles uroporphyrinogen III and uroporphyrinogen I. The former is the precursor of all the natural porphyrins and chlorins. The formation of uroporphyrinogen III is catalysed by a dual enzymic system, porphobilinogen deaminase and uroporphyrinogen III cosynthase. Deaminase polymerizes four porphobilinogen units on the enzymic surface, without liberation of free intermediates into the reaction medium, and forms uroporphyrinogen I. Cosynthase enters into association with the deaminase, and acts as a 'specifier protein' of the latter, changing the mode of porphobilinogen condensation on the enzymic surface. The association is independent of the presence of substrate. While deaminase catalyses the head-to-tail condensation of the porphobilinogen units, the association deaminase-cosynthase catalyses the head-to-head condensation of the same units. As a result different enzyme-bound dipyrrylmethanes are formed form the beginning of the process, and this can be demonstrated by using synthetic dipyrrylmethanes and tripyrranes.     

Delta-aminolevulinic acid dehydratase from Plasmodium falciparum: indigenous versus imported

The heme biosynthetic pathway of the malaria parasite is a drug target and the import of host delta-aminolevulinate dehydratase (ALAD), the second enzyme of the pathway, from the red cell cytoplasm by the intra erythrocytic malaria parasite has been demonstrated earlier in this laboratory. In this study, ALAD encoded by the Plasmodium falciparum genome (PfALAD) has been cloned, the protein overexpressed in Escherichia coli, and then characterized. The mature recombinant enzyme (rPfALAD) is enzymatically active and behaves as an octamer with a subunit Mr of 46,000. The enzyme has an alkaline pH optimum of 8.0 to 9.0. rPfALAD does not require any metal ion for activity, although it is stimulated by 20-30% upon addition of Mg2+. The enzyme is inhibited by Zn2+ and succinylacetone. The presence of PfALAD in P. falciparum can be demonstrated by Western blot analysis and immunoelectron microscopy. The enzyme has been localized to the apicoplast of the malaria parasite. Homology modeling studies reveal that PfALAD is very similar to the enzyme species from Pseudomonas aeruginosa, but manifests features that are unique and different from plant ALADs as well as from those of the bacterium. It is concluded that PfALAD, while resembling plant ALADs in terms of its alkaline pH optimum and apicoplast localization, differs in its Mg2+ independence for catalytic activity or octamer stabilization. Expression levels of PfALAD in P. falciparum, based on Western blot analysis, immunoelectron microscopy, and EDTA-resistant enzyme activity assay reveals that it may account for about 10% of the total ALAD activity in the parasite, the rest being accounted for by the host enzyme imported by the parasite. It is proposed that the role of PfALAD may be confined to heme synthesis in the apicoplast that may not account for the total de novo heme biosynthesis in the parasite.     

Uroporphyrinogen synthase in human blood: developmental studies.

Uroporphyrinogen synthase (URO-S), the enzyme that catalyzes the conversion of porphobilinogen to uroporphyrinogen I, has been measured in whole blood lysates by a fluorometric microassay. Cord and fetal bloods have 3 and 6 times the specific activity, respectively, of adult control subjects. The three groups seem to present a similar genetic heterogenity with ratios of highest to lowest URO-S specific activity close to 2. These results establish normal ranges for URO-S activity in human blood, which may be useful for the early detection of carriers of a gene for acute intermittent porphyria.     

Biosynthesis of uroporphyrinogens. Interaction among 2-aminomethyltripyrranes and the enzymatic system

Many hypotheses on uroporphyrinogen biosynthesis advanced the possibility that 2-aminomethyltripyrranes formed by porphobilinogen deaminase are further substrates or uroporphyrinogen III co-synthase in the presence of porphobilinogen. These proposals were put to test by employing synthetic 2-aminomethyltripyrranes formally derived from porphobilinogen. None of them was found to be by itself a substrate of deaminase or of co-synthase in the presence of porphobilinogen. The tripyrranes chemically formed uroporphyrinogens by dimerization reactions, and the latter had to be deducted in control runs during the enzymatic studies. Two of the tripyrranes examined, the 2-aminomethyltripyrrane 7 and the 2-aminomethyltripyrrane 8, were found to be incorporated into enzymatically formed uroporphyrinogen III in the presence of porphobilinogen and of the deaminase-co-synthase system. While the former gave only a slight incorporation, the latter was incorporated in about 16%. No incorporation of 8 into uroporphyrinogen I was detected. On the basis of these results, and of the previous results obtained with 2-aminomethyldipyrrylmethanes, an outline of the most likely pathway of uroporphyrinogen III biosynthesis from porphobilinogen is given.     

A molecular study of congenital erythropoietic porphyria in cattle

Previous studies have shown that congenital erythropoietic porphyria (CEP) in cattle is caused by an inherited deficiency of the enzyme uroporphyrinogen III synthase (UROS) encoded by the UROS gene. In this study, we have established the pedigree of an extended Holstein family in which the disease is segregating in a manner consistent with autosomal recessive inheritance. Biochemical analyses demonstrated accumulation of uroporphyrin, thus confirming that it is indeed insufficient activity of UROS which is the cause of the disease. We have therefore sequenced all nine exons of UROS in affected and non-affected individuals without detecting any potential causative mutations. However, a single nucleotide polymorphism (SNP) located within the spliceosome attachment region in intron 8 of UROS is shown to segregate with the disease allele. Our study supports the hypothesis that CEP in cattle is caused by a mutation affecting UROS; however, additional functional studies are needed to identify the causative mutation.     

Plastid-associated Porphobilinogen Synthase from Toxoplasma gondii: KINETIC AND STRUCTURAL PROPERTIES VALIDATE THERAPEUTIC POTENTIAL*

Apicomplexan parasites (including Plasmodium spp. and Toxoplasma gondii) employ a four-carbon pathway for de novo heme biosynthesis, but this pathway is distinct from the animal/fungal C4 pathway in that it is distributed between three compartments: the mitochondrion, cytosol, and apicoplast, a plastid acquired by secondary endosymbiosis of an alga. Parasite porphobilinogen synthase (PBGS) resides within the apicoplast, and phylogenetic analysis indicates a plant origin. The PBGS family exhibits a complex use of metal ions (Zn²⁺ and Mg²⁺) and oligomeric states (dimers, hexamers, and octamers). Recombinant T. gondii PBGS (TgPBGS) was purified as a stable ∼320-kDa octamer, and low levels of dimers but no hexamers were also observed. The enzyme displays a broad activity peak (pH 7–8.5), with a K_m for aminolevulinic acid of ∼150 μm and specific activity of ∼24 μmol of porphobilinogen/mg of protein/h. Like the plant enzyme, TgPBGS responds to Mg²⁺ but not Zn²⁺ and shows two Mg²⁺ affinities, interpreted as tight binding at both the active and allosteric sites. Unlike other Mg²⁺-binding PBGS, however, metal ions are not required for TgPBGS octamer stability. A mutant enzyme lacking the C-terminal 13 amino acids distinguishing parasite PBGS from plant and animal enzymes purified as a dimer, suggesting that the C terminus is required for octamer stability. Parasite heme biosynthesis is inhibited (and parasites are killed) by succinylacetone, an active site-directed suicide substrate. The distinct phylogenetic, enzymatic, and structural features of apicomplexan PBGS offer scope for developing selective inhibitors of the parasite enzyme based on its quaternary structure characteristics.