Systematically testing human HMBS missense variants to reveal mechanism and pathogenic variation.

Defects in hydroxymethylbilane synthase (HMBS) can cause acute intermittent porphyria (AIP), an acute neurological disease. Although sequencing-based diagnosis can be definitive, ∼⅓ of clinical HMBS variants are missense variants, and most clinically reported HMBS missense variants are designated as "variants of uncertain significance" (VUSs). Using saturation mutagenesis, en masse selection, and sequencing, we applied a multiplexed validated assay to both the erythroid-specific and ubiquitous isoforms of HMBS, obtaining confident functional impact scores for >84% of all possible amino acid substitutions. The resulting variant effect maps generally agreed with biochemical expectations and provide further evidence that HMBS can function as a monomer. Additionally, the maps implicated specific residues as having roles in active site dynamics, which was further supported by molecular dynamics simulations. Most importantly, these maps can help discriminate pathogenic from benign HMBS variants, proactively providing evidence even for yet-to-be-observed clinical missense variants.

Developing a novel heme biosensor to produce high-active hemoproteins in Pichia pastoris through comparative transcriptomics.

The development of a heme-responsive biosensor for dynamic pathway regulation in eukaryotes has never been reported, posing a challenge for achieving the efficient synthesis of multifunctional hemoproteins and maintaining intracellular heme homeostasis. Herein, a biosensor containing a newly identified heme-responsive promoter, CRISPR/dCas9, and a degradation tag N-degron was designed and optimized to fine-tune heme biosynthesis in the efficient heme-supplying Pichia pastoris P1H9 chassis. After identifying literature-reported promoters insensitive to heme, the endogenous heme-responsive promoters were mined by transcriptomics, and an optimal biosensor was screened from different combinations of regulatory elements. The dynamic regulation pattern of the biosensor was validated by the transcriptional fluctuations of the HEM2 gene involved in heme biosynthesis and the subsequent responsive changes in intracellular heme titers. We demonstrate the efficiency of this regulatory system by improving the production of high-active porcine myoglobin and soy hemoglobin, which can be used to develop artificial meat and artificial metalloenzymes. Moreover, these findings can offer valuable strategies for the synthesis of other hemoproteins.

Congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) is a rare autosomal recessive disease due to the deficient, but not absent, activity of uroporphyrinogen III synthase (UROS), the fourth enzyme in the heme biosynthesis pathway. Biallelic variants in the UROS gene result in decreased UROS enzymatic activity and the accumulation of non-physiologic Type I porphyrins in cells and fluids. Overproduced uroporphyrins in haematopoietic cells are released into the circulation and distributed to tissues, inducing primarily hematologic and dermatologic symptoms. The clinical manifestations vary in severity ranging from non-immune hydrops fetalis in utero to mild dermatologic manifestations in adults. Here, the biochemical, molecular and clinical features of CEP as well as current and new treatment options, including the rescue of UROS enzyme activity by chaperones, are presented.

Case-based discussion of the acute hepatic porphyrias: Updates on pathogenesis, diagnosis and management.

The acute hepatic porphyrias (AHPs) include three autosomal dominant disorders, acute intermittent porphyria, variegate porphyria  and hereditary coproporphyria, and the ultra-rare autosomal recessive 5-aminolevulinic acid dehydratase-deficient porphyria. All four are characterized by episodic acute neurovisceral attacks that can be life-threatening if left untreated. The attacks are precipitated by factors that induce hepatic 5-aminolevulinic acid synthase 1 (ALAS1), resulting in accumulation of the porphyrin precursors, 5-aminolevulinic acid and porphobilinogen, which are believed to cause neurotoxicity. Diagnosis of these rare disorders is often delayed because the symptoms are non-specific with many common aetiologies. However, once clinical suspicion of an AHP is raised, diagnosis can be made by specialized biochemical testing, particularly during attacks. Moderate or severe attacks are treated with intravenous hemin infusions, together with supportive care to relieve pain and other symptoms. Prophylactic treatments are recommended in patients with confirmed recurrent attacks (≥4 attacks in a maximum period of 12 months), the most effective being givosiran, an RNAi therapeutic targeting hepatocyte ALAS1 mRNA. AHP patients with clinically and/or biochemically active disease are at elevated risk for developing long-term complications, including chronic kidney disease, chronic hypertension and hepatocellular carcinoma, thus, surveillance is recommended. Here, using a case-based format, we provide an update on the pathogenesis, diagnosis and treatment of the AHPs based on literature review and clinical experiences.

Improved biosynthesis of heme in Bacillus subtilis through metabolic engineering assisted fed-batch fermentation.

BACKGROUND: Heme is an iron/porphyrin complex compound, widely used in the health care, food, and pharmaceutical industries. It is more advantageous and attractive to develop microbial cell factories to produce heme by fermentation, with lower production costs and environmentally more friendly procedures than those of the traditional extraction based on animal blood. In this study, Bacillus subtilis, a typical industrial model microorganism of food safety grade, was used for the first time as the host to synthesize heme. RESULTS: The heme biosynthetic pathway was engineered as four modules, the endogenous C5 pathway, the heterologous C4 pathway, the uroporphyrinogen (urogen) III synthesis pathway, and the downstream synthesis pathway. Knockout of hemX encoding the negative effector of the concentration of HemA, overexpression of hemA encoding glutamyl-tRNA reductase, and knockout of rocG encoding the major glutamate dehydrogenase in the C5 pathway, resulted in an increase of 427% in heme production. Introduction of the heterologous C4 pathway showed a negligible effect on heme biosynthesis. Overexpression of hemCDB, which encoded hydroxymethylbilane synthase, urogen III synthase, and porphobilinogen synthase participating in the urogen III synthesis pathway, increased heme production by 39%. Knockouts of uroporphyrinogen methyltransferase gene nasF and both heme monooxygenase genes hmoA and hmoB in the downstream synthesis pathway increased heme production by 52%. The engineered B. subtilis produced 248.26 ± 6.97 mg/L of total heme with 221.83 ± 4.71 mg/L of extracellular heme during the fed-batch fermentation in 10 L fermenter. CONCLUSIONS: Strengthening endogenous C5 pathway, urogen III synthesis pathway and downstream synthesis pathway promoted the biosynthesis of heme in B. subtilis. The engineered B. subtilis strain has great potential as a microbial cell factory for efficient industrial heme production.

[Characteristics of δ-Aminolevulinic Acid Dehydratase of the Cold-Water Sponge Halisarca dujardinii].

δ-Aminolevulinic acid dehydratase (ALAD) is a key enzyme of the cytoplasmic heme biosynthesis pathway. The primary structure of the ALAD gene, the multimeric structure of the ALAD/hemB protein, and ALAD expression during the annual reproductive cycle were studied in the cold-water marine sponge Halisarca dujardinii. The results implicated the GATA-1 transcription factor and DNA methylation in regulating ALAD expression. Re-aggregation of sponge cells was accompanied by a decrease in ALAD expression and a change in the cell content of an active ALAD/hemB form. Further study of heme biosynthesis and the role of ALAD/hemB in morphogenesis of basal animals may provide new opportunities for treating pathologies in higher animals.

Genomic Insights into the Origin and Evolution of Molluscan Red-Bloodedness in the Blood Clam Tegillarca granosa.

Blood clams differ from their molluscan kins by exhibiting a unique red-blood (RB) phenotype; however, the genetic basis and biochemical machinery subserving this evolutionary innovation remain unclear. As a fundamental step toward resolving this mystery, we presented the first chromosome-level genome and comprehensive transcriptomes of the blood clam Tegillarca granosa for an integrated genomic, evolutionary, and functional analyses of clam RB phenotype. We identified blood clam-specific and expanded gene families, as well as gene pathways that are of RB relevant. Clam-specific RB-related hemoglobins (Hbs) showed close phylogenetic relationships with myoglobins (Mbs) of blood clam and other molluscs without the RB phenotype, indicating that clam-specific Hbs were likely evolutionarily derived from the Mb lineage. Strikingly, similar to vertebrate Hbs, blood clam Hbs were present in a form of gene cluster. Despite the convergent evolution of Hb clusters in blood clam and vertebrates, their Hb clusters may have originated from a single ancestral Mb-like gene as evidenced by gene phylogeny and synteny analysis. A full suite of enzyme-encoding genes for heme synthesis was identified in blood clam, with prominent expression in hemolymph and resembling those in vertebrates, suggesting a convergence of both RB-related Hb and heme functions in vertebrates and blood clam. RNA interference experiments confirmed the functional roles of Hbs and key enzyme of heme synthesis in the maintenance of clam RB phenotype. The high-quality genome assembly and comprehensive transcriptomes presented herein serve new genomic resources for the super-diverse phylum Mollusca, and provide deep insights into the origin and evolution of invertebrate RB.

Bi-allelic hydroxymethylbilane synthase inactivation defines a homogenous clinico-molecular subtype of hepatocellular carcinoma.

BACKGROUND & AIMS: Acute intermittent porphyria (AIP), caused by heterozygous germline mutations of the heme synthesis pathway enzyme HMBS (hydroxymethylbilane synthase), confers a high risk of hepatocellular carcinoma (HCC) development. Yet, the role of HMBS in liver tumorigenesis remains unclear. METHODS: Herein, we explore HMBS alterations in a large series of 758 HCC cases, including 4 patients with AIP. We quantify the impact of HMBS mutations on heme biosynthesis pathway intermediates and we investigate the molecular and clinical features of HMBS-mutated tumors. RESULTS: We identify recurrent bi-allelic HMBS inactivation, both in patients with AIP acquiring a second somatic HMBS mutation and in sporadic HCC with 2 somatic hits. HMBS alterations are enriched in truncating mutations, in particular in splice regions, leading to abnormal transcript structures. Bi-allelic HMBS inactivation results in a massive accumulation of its toxic substrate porphobilinogen and synergizes with CTNNB1-activating mutations, leading to the development of well-differentiated tumors with a transcriptomic signature of Wnt/ß-catenin pathway activation and a DNA methylation signature related to ageing. HMBS-inactivated HCC mostly affects females, in the absence of fibrosis and classical HCC risk factors. CONCLUSIONS: These data identify HMBS as a tumor suppressor gene whose bi-allelic inactivation defines a homogenous clinical and molecular HCC subtype. LAY SUMMARY: Heme (the precursor to hemoglobin, which plays a key role in oxygen transport around the body) synthesis occurs in the liver and involves several enzymes including hydroxymethylbilane synthase (HMBS). HMBS mutations cause acute intermittent porphyria, a disease caused by the accumulation of toxic porphyrin precursors. Herein, we show that HMBS inactivation is also involved in the development of liver cancers with distinct clinical and molecular characteristics.

Crystal structures of hydroxymethylbilane synthase complexed with a substrate analog: a single substrate-binding site for four consecutive condensation steps.

Hydroxymethylbilane synthase (HMBS), which is involved in the heme biosynthesis pathway, has a dipyrromethane cofactor and combines four porphobilinogen (PBG) molecules to form a linear tetrapyrrole, hydroxymethylbilane. Enzyme kinetic study of human HMBS using a PBG-derivative, 2-iodoporphobilinogen (2-I-PBG), exhibited noncompetitive inhibition with the inhibition constant being 5.4 ± 0.3 µM. To elucidate the reaction mechanism of HMBS in detail, crystal structure analysis of 2-I-PBG-bound holo-HMBS and its reaction intermediate possessing two PBG molecules (ES2), and inhibitor-free ES2 was performed at 2.40, 2.31, and 1.79 Šresolution, respectively. Their overall structures are similar to that of inhibitor-free holo-HMBS, and the differences are limited near the active site. In both 2-I-PBG-bound structures, 2-I-PBG is located near the terminus of the cofactor or the tetrapyrrole chain. The propionate group of 2-I-PBG interacts with the side chain of Arg173, and its acetate group is associated with the side chains of Arg26 and Ser28. Furthermore, the aminomethyl group and pyrrole nitrogen of 2-I-PBG form hydrogen bonds with the side chains of Gln34 and Asp99, respectively. These amino acid residues form a single substrate-binding site, where each of the four PBG molecules covalently binds to the cofactor (or oligopyrrole chain) consecutively, ultimately forming a hexapyrrole chain. Molecular dynamics simulation of the ES2 intermediate suggested that the thermal fluctuation of the lid and cofactor-binding loops causes substrate recruitment and oligopyrrole chain shift needed for consecutive condensation. Finally, the hexapyrrole chain is hydrolyzed self-catalytically to produce hydroxymethylbilane.

[Anesthesia in patients with acute porphyria]. / Anästhesie bei Patienten mit akuter Porphyrie.

Porphyrias are a group of rare, mostly inherited metabolic disorders of heme biosynthesis. Each type of porphyria results from a specific deficiency of one of the pathway enzymes, causing a characteristic accumulation and excretion of heme precursors. Diagnosis is confirmed by the biochemical detection of these porphyrins and the precursors in urine, feces and blood. Porphyrias can be classified into acute and non-acute forms. The clinical presentation is unspecific and includes acute neurovisceral and/or cutaneous symptoms. The latent phase can evolve into a potentially life-threatening acute crisis, which is often misdiagnosed. The four acute hepatic porphyrias are relevant for anesthesiologists as precipitating factors are commonly found in the perioperative setting. Safe anesthetic management in cases of known porphyria is possible by adherence to current recommendations. The immediate administration of heme arginate as specific treatment for acute attacks is decisive for the outcome.

Characterization of the apicoplast-localized enzyme TgUroD in Toxoplasma gondii reveals a key role of the apicoplast in heme biosynthesis.

Apicomplexan parasites such as Toxoplasma gondii possess an unusual heme biosynthesis pathway whose enzymes localize to the mitochondrion, cytosol, or apicoplast, a nonphotosynthetic plastid present in most apicomplexans. To characterize the involvement of the apicoplast in the T. gondii heme biosynthesis pathway, we investigated the role of the apicoplast-localized enzyme uroporphyrinogen III decarboxylase (TgUroD). We found that TgUroD knockdown impaired parasite proliferation, decreased free heme levels in the parasite, and decreased the abundance of heme-containing c-type cytochrome proteins in the parasite mitochondrion. We validated the effects of heme loss on mitochondrial cytochromes by knocking down cytochrome c/c1 heme lyase 1 (TgCCHL1), a mitochondrial enzyme that catalyzes the covalent attachment of heme to c-type cytochromes. TgCCHL1 depletion reduced parasite proliferation and decreased the abundance of c-type cytochromes. We further sought to characterize the overall importance of TgUroD and TgCCHL1 for both mitochondrial and general parasite metabolism. TgUroD depletion decreased cellular ATP levels, mitochondrial oxygen consumption, and extracellular acidification rates. By contrast, depletion of TgCCHL1 neither diminished ATP levels in the parasite nor impaired extracellular acidification rate, but resulted in specific defects in mitochondrial oxygen consumption. Together, our results indicate that the apicoplast has a key role in heme biology in T. gondii and is important for both mitochondrial and general parasite metabolism. Our study highlights the importance of heme and its synthesis in these parasites.

Animal-free heme production for artificial meat in Corynebacterium glutamicum via systems metabolic and membrane engineering.

Recently, heme has attracted much attention as a main ingredient that mimics meat flavor in artificial meat in the food industry. Here, we developed Corynebacterium glutamicum capable of high-yield production of heme with systems metabolic engineering and modification of membrane surface. The combination of two precursor pathways based on thermodynamic information increased carbon flux toward heme and porphyrin intermediate biosynthesis. The co-overexpression of genes involved in a noncanonical downstream pathway and the gene encoding the transcriptional regulator DtxR significantly enhanced heme production. The overexpression of the putative heme exporters, knockout of heme-binding proteins, modification of the cell wall by chemical treatment, and reduction of intermediate UP III substantially improved heme secretion. The fed-batch fermentation showed a maximum heme titer of 309.18 ± 16.43 mg l-1, including secreted heme of 242.95 ± 11.45 mg l-1, a yield on glucose of 0.61 mmol mol-1, and productivity of 6.44 mg l-1h-1, which are the highest values reported to date. These results demonstrate that engineered C. glutamicum can be an attractive cell factory for animal-free heme production.

Mutation in human CLPX elevates levels of δ-aminolevulinate synthase and protoporphyrin IX to promote erythropoietic protoporphyria.

Loss-of-function mutations in genes for heme biosynthetic enzymes can give rise to congenital porphyrias, eight forms of which have been described. The genetic penetrance of the porphyrias is clinically variable, underscoring the role of additional causative, contributing, and modifier genes. We previously discovered that the mitochondrial AAA+ unfoldase ClpX promotes heme biosynthesis by activation of δ-aminolevulinate synthase (ALAS), which catalyzes the first step of heme synthesis. CLPX has also been reported to mediate heme-induced turnover of ALAS. Here we report a dominant mutation in the ATPase active site of human CLPX, p.Gly298Asp, that results in pathological accumulation of the heme biosynthesis intermediate protoporphyrin IX (PPIX). Amassing of PPIX in erythroid cells promotes erythropoietic protoporphyria (EPP) in the affected family. The mutation in CLPX inactivates its ATPase activity, resulting in coassembly of mutant and WT protomers to form an enzyme with reduced activity. The presence of low-activity CLPX increases the posttranslational stability of ALAS, causing increased ALAS protein and ALA levels, leading to abnormal accumulation of PPIX. Our results thus identify an additional molecular mechanism underlying the development of EPP and further our understanding of the multiple mechanisms by which CLPX controls heme metabolism.

Loss of hepatocyte ß-catenin protects mice from experimental porphyria-associated liver injury.

BACKGROUND & AIMS: Porphyrias result from anomalies of heme biosynthetic enzymes and can lead to cirrhosis and hepatocellular cancer. In mice, these diseases can be modeled by administration of a diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), which causes accumulation of porphyrin intermediates, resulting in hepatobiliary injury. Wnt/ß-catenin signaling has been shown to be a modulatable target in models of biliary injury; thus, we investigated its role in DDC-driven injury. METHODS: ß-Catenin (Ctnnb1) knockout (KO) mice, Wnt co-receptor KO mice, and littermate controls were fed a DDC diet for 2 weeks. ß-Catenin was exogenously inhibited in hepatocytes by administering ß-catenin dicer-substrate RNA (DsiRNA), conjugated to a lipid nanoparticle, to mice after DDC diet and then weekly for 4 weeks. In all experiments, serum and livers were collected; livers were analyzed by histology, western blotting, and real-time PCR. Porphyrin was measured by fluorescence, quantification of polarized light images, and liquid chromatography-mass spectrometry. RESULTS: DDC-fed mice lacking ß-catenin or Wnt signaling had decreased liver injury compared to controls. Exogenous mice that underwent ß-catenin suppression by DsiRNA during DDC feeding also showed less injury compared to control mice receiving lipid nanoparticles. Control livers contained extensive porphyrin deposits which were largely absent in mice lacking ß-catenin signaling. Notably, we identified a network of key heme biosynthesis enzymes that are suppressed in the absence of ß-catenin, preventing accumulation of toxic protoporphyrins. Additionally, mice lacking ß-catenin exhibited fewer protein aggregates, improved proteasomal activity, and reduced induction of autophagy, all contributing to protection from injury. CONCLUSIONS: ß-Catenin inhibition, through its pleiotropic effects on metabolism, cell stress, and autophagy, represents a novel therapeutic approach for patients with porphyria. LAY SUMMARY: Porphyrias are disorders resulting from abnormalities in the steps that lead to heme production, which cause build-up of toxic by-products called porphyrins. Liver is commonly either a source or a target of excess porphyrins, and complications can range from minor abnormalities to liver failure. In this report, we inhibited Wnt/ß-catenin signaling in an experimental model of porphyria, which resulted in decreased liver injury. Targeting ß-catenin affected multiple components of the heme biosynthesis pathway, thus preventing build-up of porphyrin intermediates. Our study suggests that drugs inhibiting ß-catenin activity could reduce the amount of porphyrin accumulation and help alleviate symptoms in patients with porphyria.

Erythropoietic Protoporphyria and X-Linked Protoporphyria: pathophysiology, genetics, clinical manifestations, and management.

Erythropoietic Protoporphyria (EPP) and X-linked Protoporphyria (XLP) are rare, genetic photodermatoses resulting from defects in enzymes of the heme-biosynthetic pathway. EPP results from the partial deficiency of ferrochelatase, and XLP results from gain-of-function mutations in erythroid specific ALAS2. Both disorders result in the accumulation of erythrocyte protoporphyrin, which is released in the plasma and taken up by the liver and vascular endothelium. The accumulated protoporphyrin is activated by sunlight exposure, generating singlet oxygen radical reactions leading to tissue damage and excruciating pain. About 2-5% of patients develop clinically significant liver dysfunction due to protoporphyrin deposition in bile and/or hepatocytes which can advance to cholestatic liver failure requiring transplantation. Clinically these patients present with acute, severe, non-blistering phototoxicity within minutes of sun-exposure. Anemia is seen in about 47% of patients and about 27% of patients will develop abnormal serum aminotransferases. The diagnosis of EPP and XLP is made by detection of markedly increased erythrocyte protoporphyrin levels with a predominance of metal-free protoporphyrin. Genetic testing by sequencing the FECH or ALAS2 gene confirms the diagnosis. Treatment is limited to sun-protection and there are no currently available FDA-approved therapies for these disorders. Afamelanotide, a synthetic analogue of α-melanocyte stimulating hormone was found to increase pain-free sun exposure and improve quality of life in adults with EPP. It has been approved for use in the European Union since 2014 and is not available in the U.S. In addition to the development of effective therapeutics, future studies are needed to establish the role of iron and the risks related to the development of hepatopathy in these patients.

The mitochondrial heme metabolon: Insights into the complex(ity) of heme synthesis and distribution.

Heme is an essential cofactor in metazoans that is also toxic in its free state. Heme is synthesized by most metazoans and must be delivered to all cellular compartments for incorporation into a variety of hemoproteins. The heme biosynthesis enzymes have been proposed to exist in a metabolon, a protein complex consisting of interacting enzymes in a metabolic pathway. Metabolons enhance the function of enzymatic pathways by creating favorable microenvironments for pathway enzymes and intermediates, facilitating substrate transport, and providing a scaffold for interactions with other pathways, signaling molecules, or organelles. Herein we detail growing evidence for a mitochondrial heme metabolon and discuss its implications for the study of heme biosynthesis and cellular heme homeostasis.

Murine models of the human porphyrias: Contributions toward understanding disease pathogenesis and the development of new therapies.

Mouse models of the human porphyrias have proven useful for investigations of disease pathogenesis and to facilitate the development of new therapeutic approaches. To date, mouse models have been generated for all major porphyrias, with the exception of X-linked protoporphyria (XLP) and the ultra rare 5-aminolevulinic acid dehydratase deficient porphyria (ADP). Mouse models have been generated for the three autosomal dominant acute hepatic porphyrias, acute intermittent porphyria (AIP), hereditary coproporphyria (HCP), and variegate porphyria (VP). The AIP mice, in particular, provide a useful investigative model as they have been shown to have acute biochemical attacks when induced with the prototypic porphyrinogenic drug, phenobarbital. In addition to providing important insights into the disease pathogenesis of the neurological impairment in AIP, these mice have been valuable for preclinical evaluation of liver-targeted gene therapy and RNAi-mediated approaches. Mice with severe HMBS deficiency, which clinically and biochemically mimic the early-onset homozygous dominant AIP (HD-AIP) patients, have been generated and were used to elucidate the striking phenotypic differences between AIP and HD-AIP. Mice modeling the hepatocutaneous porphyria, porphyria cutanea tarda (PCT), made possible the identification of the iron-dependent inhibitory mechanism of uroporphyrinogen decarboxylase (UROD) that leads to symptomatic PCT. Mouse models for the two autosomal recessive erythropoietic porphyrias, congenital erythropoietic porphyria (CEP) and erythropoeitic protoporphyria (EPP), recapitulate many of the clinical and biochemical features of the severe human diseases and have been particularly useful for evaluation of bone marrow transplantation and hematopoietic stem cell (HSC)-based gene therapy approaches. The EPP mice have also provided valuable insights into the underlying pathogenesis of EPP-induced liver damage and anemia.

Identification and characterization of 40 novel hydroxymethylbilane synthase mutations that cause acute intermittent porphyria.

Acute intermittent porphyria (AIP), an autosomal dominant disorder due to the half-normal activity of hydroxymethylbilane synthase (HMBS), is characterized by acute neurovisceral attacks that are precipitated by factors that induce heme biosynthesis. Molecular diagnosis is the most sensitive and specific diagnostic test for AIP, and importantly, it permits the identification of asymptomatic family members for genetic counseling and avoidance of precipitating factors. Here, we report the identification of 40 novel HMBS mutations, including 11 missense, four nonsense, 16 small insertions or deletions, eight consensus splice site mutations, and a complex insertion-deletion mutation in unrelated individuals with AIP. Prokaryotic expression of the missense mutations demonstrated that all mutants had ≤5% of expressed wildtype activity, except for c.1039G>C (p.A347P), which had 51% residual HMBS activity but was markedly thermolabile. Of note, the mutation c.612G>T (p.Q204H) altered the last nucleotide of exon 10, which resulted in an alternative HMBS transcript with an in-frame nine base-pair deletion at the 3'-terminus of exon 10 (encoding protein Q204HΔ3). When expressed, Q204HΔ3 and an in-frame three base-pair deletion (c.639_641delTGC) had no detectable HMBS activity. Western blot analyses and mapping of the missense mutations on the human HMBS crystal structure revealed that mutations near the active site or at the dimerization interface resulted in stably expressed proteins, while most that altered surface residues resulted in unstable proteins, presumably due to improper protein folding. These studies identified novel pathogenic HMBS mutations and expanded the molecular heterogeneity of AIP.

Coproheme decarboxylases - Phylogenetic prediction versus biochemical experiments.

Coproheme decarboxylases (ChdCs) are enzymes responsible for the catalysis of the terminal step in the coproporphyrin-dependent heme biosynthesis pathway. Phylogenetic analyses confirm that the gene encoding for ChdCs is widespread throughout the bacterial world. It is found in monoderm bacteria (Firmicutes, Actinobacteria), diderm bacteria (e. g. Nitrospirae) and also in Archaea. In order to test phylogenetic prediction ChdC representatives from all clades were expressed and examined for their coproheme decarboxylase activity. Based on available biochemical data and phylogenetic analyses a sequence motif (-Y-P-M/F-X-K/R-) is defined for ChdCs. We show for the first time that in diderm bacteria an active coproheme decarboxylase is present and that the archaeal ChdC homolog from Sulfolobus solfataricus is inactive and its physiological role remains elusive. This shows the limitation of phylogenetic prediction of an enzymatic activity, since the identified sequence motif is equally conserved across all previously defined clades.

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.