Alkaptonuria in Russia: mutational spectrum and novel variants.

Alkaptonuria is a rare genetic disease caused by mutations in HGD gene. Here we report the results of genetic and biochemical analysis of 49 Russian patients with alkaptonuria. One of the common variants c.481G > A; p.(Gly161Arg) comprising 72.4% of identified alleles was found in 45 of 49 patients in our cohort, which is probably the highest frequency of this variant worldwide. 9 novel variants were found: 6 missense, 2 splicing and 1 loss of start-codon. For missense variants we performed bioinformatic analysis, protein 3D-modeling and molecular dynamics simulations, which strongly suggest their pathogenic effect. For the rare synonymous variant c.753C > T; p.(Gly251Gly), which was found in 3 cases and predicted to activate cryptic splice site, we performed the detailed functional analysis on patient's cDNA and minigene assay and confirmed its pathogenicity.

Transient pockets as mediators of gas molecules routes inside proteins: The case study of dioxygen pathway in homogentisate 1,2-dioxygenase and its implication in Alkaptonuria development.

Alkaptonuria (AKU) is an ultra-rare disease caused by mutations in homogentisate 1,2-dioxygenase (HGD) enzyme, characterized by the loss of enzymatic activity and the accumulation of its substrate, homogentisic acid (HGA) in different tissues, leading to ochronosis and organ degeneration. Although the pathological effects of HGD mutations are largely studied, less is known about the structure of the enzyme, in particular the pathways for dioxygen diffusion to the active site, required for the enzymatic reaction, are still uninvestigated. In the present project, the combination of two in silico techniques, Molecular Dynamics (MD) simulation and Implicit Ligand Sampling (ILS), was used to delineate gas diffusion routes in HGD enzyme. A route from the central opening of the hexameric structure of the enzyme to the back of the active site trough the protein moiety was identified as the path for dioxygen diffusion, also overlapping with a transient pocket, which then assumes an important role in dioxygen diffusion. Along the route the sequence location of the missense variant E401Q, responsible for AKU development, was also found, suggesting such mutation to be conducive of enzymatic activity loss by altering the flow dynamics of dioxygen. Our in silico approach allowed also to delineate the route of HGA substrate to the active site, until now only supposed.

Catechol 1,2-Dioxygenase is an Analogue of Homogentisate 1,2-Dioxygenase in Pseudomonas chlororaphis Strain UFB2.

Catechol dioxygenases in microorganisms cleave catechol into cis-cis-muconic acid or 2-hydroxymuconic semialdehyde via the ortho- or meta-pathways, respectively. The aim of this study was to purify, characterize, and predict the template-based three-dimensional structure of catechol 1,2-dioxygenase (C12O) from indigenous Pseudomonas chlororaphis strain UFB2 (PcUFB2). Preliminary studies showed that PcUFB2 could degrade 40 ppm of 2,4-dichlorophenol (2,4-DCP). The crude cell extract showed 10.34 U/mL of C12O activity with a specific activity of 2.23 U/mg of protein. A 35 kDa protein was purified to 1.5-fold with total yield of 13.02% by applying anion exchange and gel filtration chromatography. The enzyme was optimally active at pH 7.5 and a temperature of 30 °C. The Lineweaver⁻Burk plot showed the vmax and Km values of 16.67 µM/min and 35.76 µM, respectively. ES-MS spectra of tryptic digested SDS-PAGE band and bioinformatics studies revealed that C12O shared 81% homology with homogentisate 1,2-dioxygenase reported in other Pseudomonas chlororaphis strains. The characterization and optimization of C12O activity can assist in understanding the 2,4-DCP metabolic pathway in PcUFB2 and its possible application in bioremediation strategies.

Toward a generalized computational workflow for exploiting transient pockets as new targets for small molecule stabilizers: Application to the homogentisate 1,2-dioxygenase mutants at the base of rare disease Alkaptonuria.

Alkaptonuria (AKU) is an inborn error of metabolism where mutation of homogentisate 1,2-dioxygenase (HGD) gene leads to a deleterious or misfolded product with subsequent loss of enzymatic degradation of homogentisic acid (HGA) whose accumulation in tissues causes ochronosis and degeneration. There is no licensed therapy for AKU. Many missense mutations have been individuated as responsible for quaternary structure disruption of the native hexameric HGD. A new approach to the treatment of AKU is here proposed aiming to totally or partially rescue enzyme activity by targeting of HGD with pharmacological chaperones, i.e. small molecules helping structural stability. Co-factor pockets from oligomeric proteins have already been successfully exploited as targets for such a strategy, but no similar sites are present at HGD surface; hence, transient pockets are here proposed as a target for pharmacological chaperones. Transient pockets are detected along the molecular dynamics trajectory of the protein and filtered down to a set of suitable sites for structural stabilization by mean of biochemical and pharmacological criteria. The result is a computational workflow relevant to other inborn errors of metabolism requiring rescue of oligomeric, misfolded enzymes.

Insights into the Reaction Mechanism of Aromatic Ring Cleavage by Homogentisate Dioxygenase: A Quantum Mechanical/Molecular Mechanical Study.

To elucidate the reaction mechanism of the ring cleavage of homogentisate by homogentisate dioxygenase, quantum mechanical/molecular mechanical (QM/MM) calculations were carried out by using two systems in different protonation states of the substrate C2 hydroxyl group. When the substrate C2 hydroxyl group is ionized (the ionized pathway), the superoxo attack on the substrate is the rate-limiting step in the catalytic cycle, with a barrier of 15.9 kcal/mol. Glu396 was found to play an important role in stabilizing the bridge species and its O-O cleavage product by donating a proton via a hydrogen-bonded water molecule. When the substrate C2 hydroxyl group is not ionized (the nonionized pathway), the O-O bond cleavage of the bridge species is the rate-limiting step, with a barrier of 15.3 kcal/mol. The QM/MM-optimized geometries for the dioxygen and alkylperoxo complexes using the nonionized model (for the C2 hydroxyl group) are in agreement with the experimental crystal structures, suggesting that the C2 hydroxyl group is more likely to be nonionized.

Twelve novel HGD gene variants identified in 99 alkaptonuria patients: focus on 'black bone disease' in Italy.

Alkaptonuria (AKU) is an autosomal recessive disorder caused by mutations in homogentisate-1,2-dioxygenase (HGD) gene leading to the deficiency of HGD enzyme activity. The DevelopAKUre project is underway to test nitisinone as a specific treatment to counteract this derangement of the phenylalanine-tyrosine catabolic pathway. We analysed DNA of 40 AKU patients enrolled for SONIA1, the first study in DevelopAKUre, and of 59 other AKU patients sent to our laboratory for molecular diagnostics. We identified 12 novel DNA variants: one was identified in patients from Brazil (c.557T>A), Slovakia (c.500C>T) and France (c.440T>C), three in patients from India (c.469+6T>C, c.650-85A>G, c.158G>A), and six in patients from Italy (c.742A>G, c.614G>A, c.1057A>C, c.752G>A, c.119A>C, c.926G>T). Thus, the total number of potential AKU-causing variants found in 380 patients reported in the HGD mutation database is now 129. Using mCSM and DUET, computational approaches based on the protein 3D structure, the novel missense variants are predicted to affect the activity of the enzyme by three mechanisms: decrease of stability of individual protomers, disruption of protomer-protomer interactions or modification of residues in the region of the active site. We also present an overview of AKU in Italy, where so far about 60 AKU cases are known and DNA analysis has been reported for 34 of them. In this rather small group, 26 different HGD variants affecting function were described, indicating rather high heterogeneity. Twelve of these variants seem to be specific for Italy.

A new formulation of protein evolutionary models that account for structural constraints.

Despite the importance of a thermodynamically stable structure with a conserved fold for protein function, almost all evolutionary models neglect site-site correlations that arise from physical interactions between neighboring amino acid sites. This is mainly due to the difficulty in formulating a computationally tractable model since rate matrices can no longer be used. Here, we introduce a general framework, based on factor graphs, for constructing probabilistic models of protein evolution with site interdependence. Conveniently, efficient approximate inference algorithms, such as Belief Propagation, can be used to calculate likelihoods for these models. We fit an amino acid substitution model of this type that accounts for both solvent accessibility and site-site correlations. Comparisons of the new model with rate matrix models and alternative structure-dependent models demonstrate that it better fits the sequence data. We also examine evolution within a family of homohexameric enzymes and find that site-site correlations between most contacting subunits contribute to a higher likelihood. In addition, we show that the new substitution model has a similar mathematical form to the one introduced in Rodrigue et al. (Rodrigue N, Lartillot N, Bryant D, Philippe H. 2005. Site interdependence attributed to tertiary structure in amino acid sequence evolution. Gene 347:207-217), although with different parameter interpretations and values. We also perform a statistical analysis of the effects of amino acids at neighboring sites on substitution probabilities and find a significant perturbation of most probabilities, further supporting the significant role of site-site interactions in protein evolution and motivating the development of new evolutionary models similar to the one described here. Finally, we discuss possible extensions and applications of the new substitution model.

Visualizing the substrate-, superoxo-, alkylperoxo-, and product-bound states at the nonheme Fe(II) site of homogentisate dioxygenase.

Homogentisate 1,2-dioxygenase (HGDO) uses a mononuclear nonheme Fe(2+) to catalyze the oxidative ring cleavage in the degradation of Tyr and Phe by producing maleylacetoacetate from homogentisate (2,5-dihydroxyphenylacetate). Here, we report three crystal structures of HGDO, revealing five different steps in its reaction cycle at 1.7-1.98 Å resolution. The resting state structure displays an octahedral coordination for Fe(2+) with two histidine residues (His331 and His367), a bidentate carboxylate ligand (Glu337), and two water molecules. Homogentisate binds as a monodentate ligand to Fe(2+), and its interaction with Tyr346 invokes the folding of a loop over the active site, effectively shielding it from solvent. Binding of homogentisate is driven by enthalpy and is entropically disfavored as shown by anoxic isothermal titration calorimetry. Three different reaction cycle intermediates have been trapped in different HGDO subunits of a single crystal showing the influence of crystal packing interactions on the course of enzymatic reactions. The observed superoxo:semiquinone-, alkylperoxo-, and product-bound intermediates have been resolved in a crystal grown anoxically with homogentisate, which was subsequently incubated with dioxygen. We demonstrate that, despite different folds, active site architectures, and Fe(2+) coordination, extradiol dioxygenases can proceed through the same principal reaction intermediates to catalyze the O2-dependent cleavage of aromatic rings. Thus, convergent evolution of nonhomologous enzymes using the 2-His-1-carboxylate facial triad motif developed different solutions to stabilize closely related intermediates in unlike environments.

First report of HGD mutations in a Chinese with alkaptonuria.

Alkaptonuria (AKU) is one of the first prototypic inborn errors in metabolism and the first human disease found to be transmitted via Mendelian autosomal recessive inheritance. It is caused by HGD mutations, which leads to a deficiency in homogentisate 1,2-dioxygenase (HGD) activity. To date, several HGD mutations have been identified as the cause of the prototypic disease across different ethnic populations worldwide. However, in Asia, the HGD mutation is very rarely reported. For the Chinese population, no literature on HGD mutation screening is available to date. In this paper, we describe two novel HGD mutations in a Chinese AKU family, the splicing mutation of IVS7+1G>C, a donor splice site of exon 7, and a missense mutation of F329C in exon 12. The predicted new splicing site of the mutated exon 7 sequence demonstrated a 303bp extension after the mutation site. The F329C mutation most probably disturbed the stability of the conformation of the two loops critical to the Fe(2+) active site of the HGD enzyme.

A novel missense HGD gene mutation, K57N, in a patient with alkaptonuria.

Alkaptonuria is a rare recessive disorder of phenylalanine/tyrosine metabolism due to a defect in the enzyme homogentisate 1,2-dioxygenase (HGD) caused by mutations in the HGD gene. We report the case of a 38 year-old male with known alkaptonuria who was referred to an adult metabolic clinic after initially presenting to an emergency department with renal colic and subsequently passing black ureteric calculi. He complained of severe debilitating lower back pain, worsening over the last few years. A CT scan revealed marked degenerative changes and severe narrowing of the disc spaces along the entire lumbar spine. Sequencing of the HGD gene revealed that he was a compound heterozygote for a previously described missense mutation in exon 13 (G360R) and a novel missense mutation in exon 3 (K57N). Lys(57) is conserved among species and mutation of this residue is predicted to affect HGD protein function by interfering with substrate traffic at the active site. In summary, we describe an alkaptonuric patient and report a novel missense HGD mutation, K57N.

Catalytic reaction mechanism of homogentisate dioxygenase: a hybrid DFT study.

Human homogentisate dioxygenase is an Fe(II)-dependent enzyme responsible for aromatic ring cleavage. The mechanism of its catalytic reaction has been investigated with the hybrid density functional method B3LYP. A relatively big model of the active site was first used to determine the substrate binding mode. It was found that binding of the substrate dianion with a vacant position trans to Glu341 is most favorable. The model was then truncated to include only the most relevant parts of the active-site residues involved in iron coordination and substrate binding. Thus, methylimidazole was used to model His292, His335, His365, and His371, while propionate modeled Glu341. The computational results suggest that the catalytic reaction of homogentisate dioxygenases involves three major chemical steps: formation of the peroxo intermediate, homolytic cleavage of the O-O bond leading to an arene oxide radical, and finally, cleavage of the six-membered ring. Calculated barriers for alternative reaction paths are markedly higher than for the proposed mechanism, and thus the computational results successfully explain the product specificity of the enzyme. Interestingly, the results indicate that the type of ring scission, intra or extra with respect to the substituents coordinating to iron, is controlled by the barrier heights for the decay of the arene oxide radical intermediate.