ABSTRACT
Congenital erythropoietic porphyria is a rare autosomal recessive disease produced by deficient activity of uroporphyrinogen III synthase, the fourth enzyme in the heme biosynthetic pathway. The disease affects many organs, can be life-threatening, and currently lacks curative treatments. Inherited mutations most commonly reduce the enzyme's stability, altering its homeostasis and ultimately blunting intracellular heme production. This results in uroporphyrin by-product accumulation in the body, aggravating associated pathological symptoms such as skin photosensitivity and disfiguring phototoxic cutaneous lesions. We demonstrated that the synthetic marketed antifungal ciclopirox binds to the enzyme, stabilizing it. Ciclopirox targeted the enzyme at an allosteric site distant from the active center and did not affect the enzyme's catalytic role. The drug restored enzymatic activity in vitro and ex vivo and was able to alleviate most clinical symptoms of congenital erythropoietic porphyria in a genetic mouse model of the disease at subtoxic concentrations. Our findings establish a possible line of therapeutic intervention against congenital erythropoietic porphyria, which is potentially applicable to most of deleterious missense mutations causing this devastating disease.
Subject(s)
Ciclopirox/therapeutic use , Drug Repositioning , Porphyria, Erythropoietic/drug therapy , Allosteric Site , Animals , Biophysical Phenomena , Cell Line , Ciclopirox/pharmacokinetics , Disease Models, Animal , Homeostasis , Mice , Phenotype , Porphyria, Erythropoietic/enzymology , Porphyria, Erythropoietic/pathology , Uroporphyrinogen III Synthetase/antagonists & inhibitors , Uroporphyrinogen III Synthetase/chemistry , Uroporphyrinogen III Synthetase/metabolismABSTRACT
There are numerous applications that use the structures of protein-ligand complexes from the PDB, such as 3D pharmacophore identification, virtual screening, and fragment-based drug design. The structures underlying these applications are potentially much more informative if they contain biologically relevant bound ligands, with high similarity to the cognate ligands. We present a study of ligand-enzyme complexes that compares the similarity of bound and cognate ligands, enabling the best matches to be identified. We calculate the molecular similarity scores using a method called PARITY (proportion of atoms residing in identical topology), which can conveniently be combined to give a similarity score for all cognate reactants or products in the reaction. Thus, we generate a rank-ordered list of related PDB structures, according to the biological similarity of the ligands bound in the structures.
Subject(s)
Acetylcholine/chemistry , Acetylcholinesterase/chemistry , Biosimilar Pharmaceuticals/chemistry , Uroporphyrinogen III Synthetase/chemistry , Uroporphyrinogens/chemistry , Acetylcholine/metabolism , Acetylcholinesterase/metabolism , Binding Sites , Biosimilar Pharmaceuticals/metabolism , Humans , Ligands , Molecular Docking Simulation , Protein Binding , Substrate Specificity , Uroporphyrinogen III Synthetase/metabolism , Uroporphyrinogens/metabolismABSTRACT
Congenital erythropoietic porphyria (CEP) is an inborn error of heme biosynthesis characterized by uroporphyrinogen III synthase (UROS) deficiency resulting in deleterious porphyrin accumulation in blood cells responsible for hemolytic anemia and cutaneous photosensitivity. We analyzed here the molecular basis of UROS impairment associated with twenty nine UROS missense mutations actually described in CEP patients. Using a computational and biophysical joint approach we predicted that most disease-causing mutations would affect UROS folding and stability. Through the analysis of enhanced green fluorescent protein-tagged versions of UROS enzyme we experimentally confirmed these data and showed that thermodynamic instability and premature protein degradation is a major mechanism accounting for the enzymatic deficiency associated with twenty UROS mutants in human cells. Since the intracellular loss in protein homeostasis is in excellent agreement with the in vitro destabilization, we used molecular dynamic simulation to rely structural 3D modification with UROS disability. We found that destabilizing mutations could be clustered within three types of mechanism according to side chain rearrangements or contact alterations within the pathogenic UROS enzyme so that the severity degree correlated with cellular protein instability. Furthermore, proteasome inhibition using bortezomib, a clinically available drug, significantly enhanced proteostasis of each unstable UROS mutant. Finally, we show evidence that abnormal protein homeostasis is a prevalent mechanism responsible for UROS deficiency and that modulators of UROS proteolysis such as proteasome inhibitors or chemical chaperones may represent an attractive therapeutic option to reduce porphyrin accumulation and prevent skin photosensitivity in CEP patients when the genotype includes a missense variant.
Subject(s)
Mutation, Missense/genetics , Porphyria, Erythropoietic/genetics , Structure-Activity Relationship , Uroporphyrinogen III Synthetase/genetics , Computational Biology , Homeostasis , Humans , Porphyria, Erythropoietic/metabolism , Porphyria, Erythropoietic/pathology , Proteasome Endopeptidase Complex/drug effects , Proteasome Endopeptidase Complex/genetics , Proteasome Inhibitors/chemistry , Proteasome Inhibitors/therapeutic use , Protein Folding , Uroporphyrinogen III Synthetase/chemistryABSTRACT
In an earlier study, we showed that two-domain segment-swapped proteins can evolve by domain swapping and fusion, resulting in a protein with two linkers connecting its domains. We proposed that a potential evolutionary advantage of this topology may be the restriction of interdomain motions, which may facilitate domain closure by a hinge-like movement, crucial for the function of many enzymes. Here, we test this hypothesis computationally on uroporphyrinogen III synthase, a two-domain segment-swapped enzyme essential in porphyrin metabolism. To compare the interdomain flexibility between the wild-type, segment-swapped enzyme (having two interdomain linkers) and circular permutants of the same enzyme having only one interdomain linker, we performed geometric and molecular dynamics simulations for these species in their ligand-free and ligand-bound forms. We find that in the ligand-free form, interdomain motions in the wild-type enzyme are significantly more restricted than they would be with only one interdomain linker, while the flexibility difference is negligible in the ligand-bound form. We also estimated the entropy costs of ligand binding associated with the interdomain motions, and find that the change in domain connectivity due to segment swapping results in a reduction of this entropy cost, corresponding to â¼20% of the total ligand binding free energy. In addition, the restriction of interdomain motions may also help the functional domain-closure motion required for catalysis. This suggests that the evolution of the segment-swapped topology facilitated the evolution of enzyme function for this protein by influencing its dynamic properties. Proteins 2016; 85:46-53. © 2016 Wiley Periodicals, Inc.
Subject(s)
Bacterial Proteins/chemistry , Thermus thermophilus/chemistry , Uroporphyrinogen III Synthetase/chemistry , Uroporphyrinogens/chemistry , Biocatalysis , Entropy , Evolution, Molecular , Ligands , Molecular Dynamics Simulation , Motion , Protein Binding , Protein Domains , Protein Structure, Secondary , Thermus thermophilus/enzymologyABSTRACT
Congenital erythropoietic porphyria (CEP) results from a deficiency in uroporphyrinogen III synthase enzyme (UROIIIS) activity that ultimately stems from deleterious mutations in the uroS gene. C73 is a hotspot for these mutations and a C73R substitution, which drastically reduces the enzyme activity and stability, is found in almost one-third of all reported CEP cases. Here, we have studied the structural basis, by which mutations in this hotspot lead to UROIIIS destabilization. First, a strong interdependency is observed between the volume of the side chain at position 73 and the folded protein. Moreover, there is a correlation between the in vitro half-life of the mutated proteins and their expression levels in eukaryotic cell lines. Molecular modelling was used to rationalize the results, showing that the mutation site is coupled to the hinge region separating the two domains. Namely, mutations at position 73 modulate the inter-domain closure and ultimately affect protein stability. By incorporating residues capable of interacting with R73 to stabilize the hinge region, catalytic activity was fully restored and a moderate increase in the kinetic stability of the enzyme was observed. These results provide an unprecedented rationale for a destabilizing missense mutation and pave the way for the effective design of molecular chaperones as a therapy against CEP.
Subject(s)
Homeostasis , Porphyria, Erythropoietic/metabolism , Protein Engineering , Uroporphyrinogen III Synthetase/metabolism , Amino Acid Substitution , Catalysis , Enzyme Activation , Enzyme Stability , Humans , Intracellular Space/metabolism , Kinetics , Models, Molecular , Mutation , Porphyria, Erythropoietic/enzymology , Porphyria, Erythropoietic/genetics , Protein Conformation , Uroporphyrinogen III Synthetase/chemistry , Uroporphyrinogen III Synthetase/geneticsABSTRACT
Congenital erythropoietic porphyria (CEP) is a rare autosomal recessive disorder characterized by uroporphyrinogen III synthase (UROS) deficiency resulting in massive porphyrin accumulation in blood cells, which is responsible for hemolytic anemia and skin photosensitivity. Among the missense mutations actually described up to now in CEP patients, the C73R and the P248Q mutations lead to a profound UROS deficiency and are usually associated with a severe clinical phenotype. We previously demonstrated that the UROS(C73R) mutant protein conserves intrinsic enzymatic activity but triggers premature degradation in cellular systems that could be prevented by proteasome inhibitors. We show evidence that the reduced kinetic stability of the UROS(P248Q) mutant is also responsible for increased protein turnover in human erythroid cells. Through the analysis of EGFP-tagged versions of UROS enzyme, we demonstrate that both UROS(C73R) and UROS(P248Q) are equally destabilized in mammalian cells and targeted to the proteasomal pathway for degradation. We show that a treatment with proteasomal inhibitors, but not with lysosomal inhibitors, could rescue the expression of both EGFP-UROS mutants. Finally, in CEP mice (Uros(P248Q/P248Q)) treated with bortezomib (Velcade), a clinically approved proteasome inhibitor, we observed reduced porphyrin accumulation in circulating RBCs and urine, as well as reversion of skin photosensitivity on bortezomib treatment. These results of medical importance pave the way for pharmacologic treatment of CEP disease by preventing certain enzymatically active UROS mutants from early degradation by using proteasome inhibitors or chemical chaperones.
Subject(s)
Models, Molecular , Porphyria, Erythropoietic/drug therapy , Proteasome Inhibitors/therapeutic use , Uroporphyrinogen III Synthetase/genetics , Uroporphyrinogen III Synthetase/metabolism , Animals , Blotting, Western , Boronic Acids/pharmacology , Boronic Acids/therapeutic use , Bortezomib , Circular Dichroism , DNA Primers/genetics , Erythroid Cells/metabolism , Humans , Mice , Mutation, Missense/genetics , Porphyria, Erythropoietic/genetics , Porphyrins/blood , Porphyrins/urine , Protein Folding , Pyrazines/pharmacology , Pyrazines/therapeutic use , Real-Time Polymerase Chain Reaction , Spectrometry, Fluorescence , Uroporphyrinogen III Synthetase/chemistryABSTRACT
Congenital erythropoietic porphyria (CEP) is a rare autosomal disease ultimately related to deleterious mutations in uroporphyrinogen III synthase (UROIIIS), the fourth enzyme of the biosynthetic route of the heme group. UROIIIS catalyzes the cyclization of the linear tetrapyrrol hydroxymethylbilane (HMB), inverting the configuration in one of the aromatic rings. In the absence of the enzyme (or when ill-functioning), HMB spontaneously degrades to the by-product uroporphyrinogen I, which cannot lead to the heme group and accumulates in the body, producing some of the symptoms observed in CEP patients. In the present chapter, clinical, biochemical, and biophysical information has been compiled to provide an integrative view on the molecular basis of CEP. The high-resolution structure of UROIIIS sheds light on the enzyme reaction mechanism while thermodynamic analysis revealed that the protein is thermolabile. Pathogenic missense mutations are found throughout the primary sequence of the enzyme. All but one of these is rarely found in patients, whereas C73R is responsible for more than one-third of the reported cases. Most of the mutant proteins (C73R included) retain partial catalytic activity but the mutations often reduce the enzyme's stability. The stabilization of the protein in vivo is discussed in the context of a new line of intervention to complement existing treatments such as bone marrow transplantation and gene therapy.
Subject(s)
Porphyria, Erythropoietic/enzymology , Uroporphyrinogen III Synthetase/chemistry , Uroporphyrinogen III Synthetase/metabolism , Animals , Biocatalysis , Humans , Porphyria, Erythropoietic/therapy , Protein Conformation , Thermodynamics , Uroporphyrinogen III Synthetase/geneticsABSTRACT
Uroporphyrinogen III synthase (U3S) is one of the key enzymes in the biosynthesis of tetrapyrrole compounds. It catalyzes the cyclization of the linear hydroxymethylbilane (HMB) to uroporphyrinogen III (uro'gen III). We have determined the crystal structure of U3S from Pseudomonas syringae pv. tomato DC3000 (psU3S) at 2.5Å resolution by the single wavelength anomalous dispersion (SAD) method. Each psU3S molecule consists of two domains interlinked by a two-stranded antiparallel ß-sheet. The conformation of psU3S is different from its homologous proteins because of the flexibility of the linker between the two domains, which might be related to this enzyme's catalytic properties. Based on mutation and activity analysis, a key residue, Arg219, was found to be important for the catalytic activity of psU3S. Mutation of Arg219 to Ala caused a decrease in enzymatic activity to about 25% that of the wild type enzyme. Our results provide the structural basis and biochemical evidence to further elucidate the catalytic mechanism of U3S.
Subject(s)
Pseudomonas syringae/enzymology , Uroporphyrinogen III Synthetase/chemistry , Catalytic Domain , Crystallography, X-Ray , Protein Structure, Secondary , Uroporphyrinogen III Synthetase/geneticsABSTRACT
PURPOSE OF WORK: To clone, express and characterize uroporphyrinogen III synthase/methyltransferase gene (cobA/hemD) from Lactobacillus reuteri. Some strains of Lb. reuteri produce cobalamin (vitamin B(12)). Cobalamin biosynthesis relies on the sequential action of more than 25 enzymes in a complex metabolic pathway. We have cloned, expressed and characterized the gene in Lb. reuteri that codes for the S-adenosy L: -methionine uroprophyrinogen III methyltransferase/synthase (CobA/HemD), a key bifunctional enzyme in the biosynthesis of cobalamin and other tetrapyrrols.
Subject(s)
Limosilactobacillus reuteri/enzymology , Methyltransferases/biosynthesis , Methyltransferases/genetics , Uroporphyrinogen III Synthetase/biosynthesis , Uroporphyrinogen III Synthetase/genetics , Amino Acid Sequence , Bacterial Proteins/biosynthesis , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Base Sequence , Cloning, Molecular , Computer Simulation , Escherichia coli/genetics , Escherichia coli/metabolism , Limosilactobacillus reuteri/genetics , Methyltransferases/chemistry , Molecular Sequence Data , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Spectrometry, Fluorescence , Uroporphyrinogen III Synthetase/chemistryABSTRACT
The first feline model of human congenital erythropoietic porphyria (CEP) due to deficient uroporphyrinogen III synthase (URO-synthase) activity was identified by its characteristic clinical phenotype, and confirmed by biochemical and molecular genetic studies. The proband, an adult domestic shorthair cat, had dark-red urine and brownish discolored teeth with red fluorescence under ultraviolet light. Biochemical studies demonstrated markedly increased uroporphyrinogen I in urine and plasma (2,650- and 10,700-fold greater than wild type, respectively), whereas urinary 5-aminolevulinic acid and porphobilinogen were lower than normal. Erythrocytic URO-synthase activity was <1% of mean wild-type activity, confirming the diagnosis and distinguishing it from feline phenocopies having acute intermittent porphyria. Sequencing of the affected cat's UROS gene revealed two missense mutations, c.140C>T (p.S47F) in exon 3 and c.331G>A (p.G111S) in exon 6, both of which were homozygous, presumably owing to parental consanguinity. Neither was present in 100 normal cat alleles. Prokaryotic expression and thermostability studies of the purified monomeric wild-type, p.S47F, p.G111S, and p.S47F/G111S enzymes showed that the p.S47F enzyme had 100% of wild-type specific activity but ~50% decreased thermostability, whereas the p.G111S and p.S47F/G111S enzymes had about 60% and 20% of wild-type specific activity, respectively, and both were markedly thermolabile. Molecular modeling results indicated that the less active/less stable p.G111S enzyme was further functionally impaired by a structural interaction induced by the presence of the S47F substitution. Thus, the synergistic interaction of two rare amino acid substitutions in the URO-synthase polypeptide caused the feline model of human CEP.
Subject(s)
Cat Diseases/enzymology , Cat Diseases/genetics , Homozygote , Mutation, Missense/genetics , Porphyria, Erythropoietic/veterinary , Porphyrins/metabolism , Uroporphyrinogen III Synthetase/genetics , Animals , Cat Diseases/blood , Cat Diseases/urine , Cats , Erythrocytes/metabolism , Male , Models, Molecular , Molecular Sequence Data , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Porphyria, Erythropoietic/blood , Porphyria, Erythropoietic/enzymology , Porphyria, Erythropoietic/urine , Porphyrins/blood , Porphyrins/urine , Uroporphyrinogen III Synthetase/chemistry , Uroporphyrinogen III Synthetase/metabolismABSTRACT
Uroporphyrinogen III synthase (U3S) catalyzes the asymmetrical cyclization of a linear tetrapyrrole to form the physiologically relevant uroporphyrinogen III (uro'gen III) isomer during heme biosynthesis. Here, we report four apoenzyme and one product complex crystal structures of the Thermus thermophilus (HB27) U3S protein. The overlay of eight crystallographically unique U3S molecules reveals a huge range of conformational flexibility, including a "closed" product complex. The product, uro'gen III, binds between the two domains and is held in place by a network of hydrogen bonds between the product's side chain carboxylates and the protein's main chain amides. Interactions of the product A and B ring carboxylate side chains with both structural domains of U3S appear to dictate the relative orientation of the domains in the closed enzyme conformation and likely remain intact during catalysis. The product C and D rings are less constrained in the structure, consistent with the conformational changes required for the catalytic cyclization with inversion of D ring orientation. A conserved tyrosine residue is potentially positioned to facilitate loss of a hydroxyl from the substrate to initiate the catalytic reaction.
Subject(s)
Uroporphyrinogen III Synthetase/chemistry , Uroporphyrinogen III Synthetase/metabolism , Uroporphyrinogens/chemistry , Uroporphyrinogens/metabolism , Crystallization , Models, Molecular , Molecular Structure , Thermus thermophilus/enzymologyABSTRACT
The asymmetric cyclic tetrapyrrole uroporphyrinogen III is the common precursor of heme, chlorophyll, siroheme, and other biological tetrapyrroles. In vivo, it is synthesized from a linear symmetric precursor (hydroxymethylbilane) by uroporphyrinogen III synthase, which catalyzes the inversion of one of the four heterocyclic rings present in the substrate. Two mechanisms have been proposed to explain this puzzling ring inversion, either through sigmatropic shifts or through the direct formation of a spirocyclic pyrrolenine intermediate. We performed the first high-level quantum mechanical calculations on model systems of this enzyme to analyze these contrasting reaction mechanisms. The results allow us to discard the sigmatropic shift mechanism and suggest that the D-ring of the hydroxymethylbilane substrate binds to the enzyme in a conformation that shields its terminal portion from reacting with ring A and prevents the formation of the biologically useless uroporphyrinogen I, whose accumulation (in individuals lacking functional uroporphyrinogen III synthase) leads to severe cutaneous dermatosis.
Subject(s)
Models, Biological , Uroporphyrinogen III Synthetase/chemistry , Uroporphyrinogen III Synthetase/metabolism , Catalysis , Electrons , Models, Molecular , Molecular Structure , Spiro Compounds/chemistryABSTRACT
Uroporphyrinogen III synthase (URO-synthase) catalyzes the cyclization and D-ring isomerization of hydroxymethylbilane (HMB) to uroporphyrinogen (URO'gen) III, the cyclic tetrapyrrole and physiologic precursor of heme, chlorophyl, and corrin. The deficient activity of human URO-synthase results in the autosomal recessive cutaneous disorder, congenital erythropoietic porphyria. Mapping of the structural determinants that specify catalysis and, potentially, protein-protein interactions is lacking. To map the active site and assess the enzyme's possible interaction in a complex with hydroxymethylbilane-synthase (HMB-synthase) and/or uroporphyrinogen-decarboxylase (URO-decarboxylase) by NMR, an efficient expression and purification procedure was developed for these cytosolic enzymes of heme biosynthesis that enabled preparation of special isotopically-labeled protein samples for NMR characterization. Using an 800 MHz instrument, assignment of the URO-synthase backbone (13)C(alpha) (100%), (1)H(alpha) (99.6%), and nonproline (1)H(N) and (15)N resonances (94%) was achieved as well as 85% of the side-chain (13)C and (1)H resonances. NMR analyses of URO-synthase titrated with competitive inhibitors N(D)-methyl-1-formylbilane (NMF-bilane) or URO'gen III, revealed resonance perturbations of specific residues lining the cleft between the two major domains of URO synthase that mapped the enzyme's active site. In silico docking of the URO-synthase crystal structure with NMF-bilane and URO'gen III was consistent with the perturbation results and provided a 3D model of the enzyme-inhibitor complex. The absence of chemical shift changes in the (15)N spectrum of URO-synthase mixed with the homogeneous HMB-synthase holoenzyme or URO-decarboxylase precluded occurrence of a stable cytosolic enzyme complex.
Subject(s)
Binding Sites , Uroporphyrinogen III Synthetase/chemistry , Amino Acid Sequence , Carbon Isotopes , Computer Simulation , Humans , Hydroxymethylbilane Synthase/isolation & purification , Kinetics , Models, Molecular , Molecular Sequence Data , Nitrogen Isotopes , Nuclear Magnetic Resonance, Biomolecular , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Thermodynamics , Uroporphyrinogen Decarboxylase/isolation & purification , Uroporphyrinogen III Synthetase/antagonists & inhibitors , Uroporphyrinogen III Synthetase/isolation & purification , Uroporphyrinogens/pharmacologyABSTRACT
The structurally related tetrapyrrolic pigments are a group of natural products that participate in many of the fundamental biosynthetic and catabolic processes of living organisms. Urogen III synthase catalyzes a key step in the formation of urogen III, a common intermediate for tetrapyrrolic natural products. In the present study, we cloned, purified, and characterized His-tagged rat urogen III synthase. The mechanism of enzymatic reaction was studied through site-directed mutagenesis of eight highly conserved residues with functional side chains around the active site followed with activity tests. Lys10, Asp17, Glu68, Tyr97, Asn121, Lys147, and His173 have not been studied previously, which were found to be unessential for enzymatic reaction. Tyr168 was identified as an important residue for enzymatic reaction catalyzed by rat urogen III synthase. Molecular modeling suggests the hydroxyl group of Tyr168 side chain is 3.5 A away from the D ring, and is within hydrogen bond distance (1.9 A) with acetate side chain of the D ring.
Subject(s)
Histidine/chemistry , Liver/enzymology , Uroporphyrinogen III Synthetase/metabolism , Amino Acid Sequence , Animals , Binding Sites , Catalysis , Cloning, Molecular , Crystallography, X-Ray , Gene Library , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Conformation , Rats , Sequence Homology, Amino Acid , Substrate Specificity , Uroporphyrinogen III Synthetase/chemistry , Uroporphyrinogen III Synthetase/geneticsABSTRACT
Heme is synthesized from glycine and succinyl CoA by eight heme synthesis enzymes. Although genetic defects in any of these enzymes are known to cause severe human blood diseases, their developmental expression in mammals is unknown. In this paper, we report two zebrafish heme synthesis enzymes, uroporphyrinogen III synthase (UROS) and protoporphyrinogen oxidase (PPO) that are well conserved in comparison to their human counterparts. Both UROS and PPO formed pairs of bilateral stripes in the lateral plate mesoderm at the 15-somite stage. At 24 h post-fertilization (hpf), UROS and PPO were predominantly expressed in the intermediate cell mass (ICM) that is the major site of primitive hematopoiesis. The expression of UROS and PPO was drastically suppressed in the bloodless mutants cloche and vlad tepes/gata 1 from 15-somite to 24hpf stages, indicating that both cloche and vlad tepes/gata 1 are required for the induction and maintenance of UROS and PPO expression in the ICM.
Subject(s)
Heme/biosynthesis , Protoporphyrinogen Oxidase/genetics , Zebrafish/genetics , Animals , Base Sequence , Cloning, Molecular , DNA Primers , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , In Situ Hybridization , Protoporphyrinogen Oxidase/chemistry , Uroporphyrinogen III Synthetase/chemistry , Uroporphyrinogen III Synthetase/geneticsSubject(s)
Alphaproteobacteria/metabolism , Archaea/metabolism , Heme/biosynthesis , Alphaproteobacteria/genetics , Archaea/enzymology , Archaea/genetics , Coproporphyrinogen Oxidase/metabolism , Genome, Bacterial , Heme/chemistry , Porphyrins/metabolism , Prokaryotic Cells , Pyrroles , Tetrapyrroles , Uroporphyrinogen III Synthetase/chemistry , Uroporphyrinogen III Synthetase/geneticsABSTRACT
Uroporphyrinogen III synthase from the cyanobacterium Anacystis nidulans was overproduced in Escherichia coli and analyzed by site specific mutagenesis. Of the nine conserved amino acids altered, only a single tyrosine mutant (Y166F) showed any significant decrease in activity suggesting this residue is critical for proper substrate binding and/or catalysis.
Subject(s)
Cyanobacteria/enzymology , Tyrosine/metabolism , Uroporphyrinogen III Synthetase/chemistry , Uroporphyrinogen III Synthetase/metabolism , Amino Acid Sequence , Amino Acid Substitution , Catalysis , Chromatography, High Pressure Liquid , Conserved Sequence , Electrophoresis, Polyacrylamide Gel , Enzyme Activation/physiology , Molecular Sequence Data , Molecular Structure , Mutagenesis, Site-Directed , Structure-Activity Relationship , Tyrosine/genetics , Uroporphyrinogen III Synthetase/geneticsABSTRACT
All tetrapyrroles are synthesized through a branched pathway, and although each tetrapyrrole receives unique modifications around the ring periphery, they all share the unifying feature of a central metal ion. Each pathway maintains a unique metal ion chelatase, and several tertiary structures have been determined, including those of the protoporphyrin ferrochelatase from both human and Bacillus subtilus, and the cobalt chelatase CbiK. These enzymes exhibit strong structural similarity and appear to function by a similar mechanism. Met8p, from Saccharomyces cerevisiae, catalyses ferrochelation during the synthesis of sirohaem, and the structure reveals a novel chelatase architecture whereby both ferrochelation and NAD(+)-dependent dehydrogenation take place in a single bifunctional active site. Asp-141 appears to participate in both catalytic reactions. The final common biosynthetic step in tetrapyrrole biosynthesis is the generation of uroporphyrinogen by uroporphyrinogen III synthase, whereby the D ring of hydroxymethylbilane is flipped during ring closure to generate the asymmetrical structure of uroporphyrinogen III. The recently derived structure of uroporphyrinogen III synthase reveals a bi-lobed structure in which the active site lies between the domains.
Subject(s)
Chelating Agents , Metals , Uroporphyrinogen III Synthetase/chemistry , Bacillus subtilis/enzymology , Humans , Models, Molecular , Protein Structure, Secondary , Pyrroles/chemistry , Saccharomyces cerevisiae/enzymology , TetrapyrrolesABSTRACT
Uroporphyrinogen III synthase, U3S, the fourth enzyme in the porphyrin biosynthetic pathway, catalyzes cyclization of the linear tetrapyrrole, hydroxymethylbilane, to the macrocyclic uroporphyrino gen III, which is used in several different pathways to form heme, siroheme, chlorophyll, F(430) and vitamin B(12). U3S activity is essential in all organisms, and decreased activity in humans leads to the autosomal recessive disorder congenital erythropoetic porphyria. We have determined the crystal structure of recombinant human U3S at 1.85 A resolution. The protein folds into two alpha/beta domains connected by a beta-ladder. The active site appears to be located between the domains, and variations in relative domain positions observed between crystallographically independent molecules indicates the presence of flexibility that may be important in the catalytic cycle. Possible mechanisms of catalysis were probed by mutating each of the four invariant residues in the protein that have titratable side chains. Additionally, six other highly conserved and titratable side chains were also mutated. In no case, however, did one of these mutations abolish enzyme activity, suggesting that the mechanism does not require acid/base catalysis.