Redox Properties of Flavin in BLUF and LOV Photoreceptor Proteins from Hybrid QM/MM Molecular Dynamics Simulation.

Flavins play an important role in many oxidation and reduction processes in biological systems. For example, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are common cofactors found in enzymatic proteins that use the special redox properties of these flavin molecules for their catalytic or photoactive functions. The redox potential of the flavin is strongly affected by its (protein) environment; however, the underlying molecular interactions of this effect are still unknown. Using hybrid quantum mechanics/molecular mechanics (QM/MM) simulation techniques, we have studied the redox properties of flavin in the gas phase, aqueous solution, and two different protein environments, in particular, a BLUF and a LOV photoreceptor domain. By mapping the changes in electrostatic potential and solvent structure, we gain insight into how specific polarization of the flavin by its environment tunes the reduction potential. We find also that accurate calculation of the reduction potentials of these systems by using the hybrid QM/MM approach is hampered by a too limited sampling of the counterion configurations and by artifacts at the QM/MM boundary. We make suggestions for how these issues can be overcome.

Design of AsLOV2 domain as a carrier of light-induced dissociable FMN photosensitizer.

Flavin mononucleotide (FMN) is a highly efficient photosensitizer (PS) yielding singlet oxygen (1 O2 ). However, its 1 O2 production efficiency significantly decreases upon isoalloxazine ring encapsulation into the protein matrix in genetically encoded photosensitizers (GEPS). Reducing isoalloxazine ring interactions with surrounding amino acids by protein engineering may increase 1 O2 production efficiency GEPS, but at the same time weakened native FMN-protein interactions may cause undesirable FMN dissociation. Here, in contrast, we intentionally induce the FMN release by light-triggered sulfur oxidation of strategically placed cysteines (oxidation-prone amino acids) in the isoalloxazine-binding site due to significantly increased volume of the cysteinyl side residue(s). As a proof of concept, in three variants of the LOV2 domain of Avena sativa (AsLOV2), namely V416C, T418C, and V416C/T418C, the effective 1 O2 production strongly correlated with the efficiency of irradiation-induced FMN dissociation (wild type (WT) < V416C < T418C < V416C/T418C). This alternative approach enables us: (i) to overcome the low 1 O2 production efficiency of flavin-based GEPSs without affecting native isoalloxazine ring-protein interactions and (ii) to utilize AsLOV2, due to its inherent binding propensity to FMN, as a PS vehicle, which is released at a target by light irradiation.

Potential therapeutic efficacy of photodynamic therapy on female hormonal-dependent cancers in a hormonal simulated microenvironment.

BACKGROUND: Photodynamic Therapy (PDT) is a clinically approved cancer treatment. Sex hormones, the key drivers for the development of female hormonal dependent cancers, might affect cancer treatment. There are seldom studies to evaluate the effect of sex hormones mimicked the menstrual cycle on the PDT mediated by prodrug 5-aminolevulinic acid (ALA) and its ester derivatives to the hormonal dependent cancers. AIMS: To evaluate the efficacy of sex hormones on Hexyl-ALA-PDT in hormonal dependent cancers and the effect of the sex hormones on heme biosynthetic pathway. METHODS: Cell culture system that mimicked the fluctuation of sex hormones 17ß-estradiol (E2) and progesterone (PG) in the menstrual cycle was developed. Two pairs of hormonal-independent and hormonal dependent uterine sarcoma and breast cancer cell lines were used as cell models. Hexyl-ALA induced PpIX production and intracellular localization were examined. Key enzymes for PpIX synthesis were analysed. Hexyl-ALA-PDT mediated phototoxicity was evaluated. RESULTS: The PpIX generation was increased in the hormonal-dependent cells (28-50 %) when cultured in the hormonal microenvironment with long incubation of Hexyl-ALA for 15 and 24 h compared to that cultured without hormones; whereas only slight difference in PpIX generation in their hormonal-independent counterpart. The PpIX generation was in a time-dependent manner. The CPOX, PPOX and FECH expressions were significantly enhanced by Hexyl-ALA-PDT in uterine sarcoma cells in hormonal microenvironment. Hexyl-ALA-PDT triggered significant increase of PPOX expression in breast cancer cells in hormonal microenvironment. The Hexyl-ALA-PDT phototoxicity was enhanced by 18-40 % in cells cultured in the hormonal system in a dose-dependent manner. CONCLUSION: The PpIX generation and the efficacy of Hexyl-ALA-PDT in both uterine sarcoma and breast cancer cells was significantly enhanced by the sex hormones via cultured in the hormonal microenvironment.

A multifunctional flavoprotein monooxygenase HspB for hydroxylation and C-C cleavage of 6-hydroxy-3-succinoyl-pyridine.

Flavoprotein monooxygenases catalyze reactions, including hydroxylation and epoxidation, involved in the catabolism, detoxification, and biosynthesis of natural substrates and industrial contaminants. Among them, the 6-hydroxy-3-succinoyl-pyridine (HSP) monooxygenase (HspB) from Pseudomonas putida S16 facilitates the hydroxylation and C-C bond cleavage of the pyridine ring in nicotine. However, the mechanism for biodegradation remains elusive. Here, we refined the crystal structure of HspB and elucidated the detailed mechanism behind the oxidative hydroxylation and C-C cleavage processes. Leveraging structural information about domains for binding the cofactor flavin adenine dinucleotide (FAD) and HSP substrate, we used molecular dynamics simulations and quantum/molecular mechanics calculations to demonstrate that the transfer of an oxygen atom from the reactive FAD peroxide species (C4a-hydroperoxyflavin) to the C3 atom in the HSP substrate constitutes a rate-limiting step, with a calculated reaction barrier of about 20 kcal/mol. Subsequently, the hydrogen atom was rebounded to the FAD cofactor, forming C4a-hydroxyflavin. The residue Cys218 then catalyzed the subsequent hydrolytic process of C-C cleavage. Our findings contribute to a deeper understanding of the versatile functions of flavoproteins in the natural transformation of pyridine and HspB in nicotine degradation.IMPORTANCEPseudomonas putida S16 plays a pivotal role in degrading nicotine, a toxic pyridine derivative that poses significant environmental challenges. This study highlights a key enzyme, HspB (6-hydroxy-3-succinoyl-pyridine monooxygenase), in breaking down nicotine through the pyrrolidine pathway. Utilizing dioxygen and a flavin adenine dinucleotide cofactor, HspB hydroxylates and cleaves the substrate's side chain. Structural analysis of the refined HspB crystal structure, combined with state-of-the-art computations, reveals its distinctive mechanism. The crucial function of Cys218 was never discovered in its homologous enzymes. Our findings not only deepen our understanding of bacterial nicotine degradation but also open avenues for applications in both environmental cleanup and pharmaceutical development.

Shining a Spotlight on Methyl Groups: Photochemically Induced Dynamic Nuclear Polarization Spectroscopy of 5-Deazariboflavin and Its Nor Analogs.

5-Deazaflavins are analogs of naturally occurring flavin cofactors. They serve as substitutes for natural flavin cofactors to investigate and modify the reaction pathways of flavoproteins. Demethylated 5-deazaflavins are potential candidates for artificial cofactors, allowing us to fine-tune the reaction kinetics and absorption characteristics of flavoproteins. In this contribution, demethylated 5-deazariboflavin radicals are investigated (1) to assess the influence of the methyl groups on the electronic structure of the 5-deazaflavin radical and (2) to explore their photophysical properties with regard to their potential as artificial cofactors. We determined the proton hyperfine structure of demethylated 5-deazariboflavins using photochemically induced dynamic nuclear polarization (photo-CIDNP) spectroscopy, as well as density functional theory (DFT). To provide context, we compare our findings to a study of flavin mononucleotide (FMN) derivatives. We found a significant influence of the methylation pattern on the absorption properties, as well as on the proton hyperfine coupling ratios of the xylene moiety, which appears to be solvent-dependent. This effect is enhanced by the replacement of N5 by C5-H in 5-deazaflavin derivatives compared to their respective flavin counterparts.

Conserved Signal Transduction Mechanisms and Dark Recovery Kinetic Tuning in the Pseudomonadaceae Short Light, Oxygen, Voltage (LOV) Protein Family.

Light-Oxygen-Voltage (LOV) flavoproteins transduce a light signal into variable signaling outputs via a structural rearrangement in the sensory core domain, which is then relayed to fused effector domains via α-helical linker elements. Short LOV proteins from Pseudomonadaceae consist of a LOV sensory core and N- and C-terminal α-helices of variable length, providing a simple model system to study the molecular mechanism of allosteric activation. Here we report the crystal structures of two LOV proteins from Pseudomonas fluorescens - SBW25-LOV in the fully light-adapted state and Pf5-LOV in the dark-state. In a comparative analysis of the Pseudomonadaceae short LOVs, the structures demonstrate light-induced rotation of the core domains and splaying of the proximal A'α and Jα helices in the N and C-termini, highlighting evidence for a conserved signal transduction mechanism. Another distinguishing feature of the Pseudomonadaceae short LOV protein family is their highly variable dark recovery, ranging from seconds to days. Understanding this variability is crucial for tuning the signaling behavior of LOV-based optogenetic tools. At 37 °C, SBW25-LOV and Pf5-LOV exhibit adduct state lifetimes of 1470 min and 3.6 min, respectively. To investigate this remarkable difference in dark recovery rates, we targeted three residues lining the solvent channel entrance to the chromophore pocket where we introduced mutations by exchanging the non-conserved amino acids from SBW25-LOV into Pf5-LOV and vice versa. Dark recovery kinetics of the resulting mutants, as well as MD simulations and solvent cavity calculations on the crystal structures suggest a correlation between solvent accessibility and adduct lifetime.

Two distinct mechanisms of flavoprotein spectral tuning revealed by low-temperature and time-dependent spectroscopy.

Flavins such as flavin mononucleotide or flavin adenine dinucleotide are bound by diverse proteins, yet have very similar spectra when in the oxidized state. Recently, we developed new variants of flavin-binding protein CagFbFP exhibiting notable blue (Q148V) or red (I52V A85Q) shifts of fluorescence emission maxima. Here, we use time-resolved and low-temperature spectroscopy to show that whereas the chromophore environment is static in Q148V, an additional protein-flavin hydrogen bond is formed upon photoexcitation in the I52V A85Q variant. Consequently, in Q148V, excitation, emission, and phosphorescence spectra are shifted, whereas in I52V A85Q, excitation and low-temperature phosphorescence spectra are relatively unchanged, while emission spectrum is altered. We also determine the x-ray structures of the two variants to reveal the flavin environment and complement the spectroscopy data. Our findings illustrate two distinct color-tuning mechanisms of flavin-binding proteins and could be helpful for the engineering of new variants with improved optical properties.

Protocatechuate hydroxylase is a novel group A flavoprotein monooxygenase with a unique substrate recognition mechanism.

Para-hydroxybenzoate hydroxylase (PHBH) is a group A flavoprotein monooxygenase that hydroxylates p-hydroxybenzoate to protocatechuate (PCA). Despite intensive studies of Pseudomonas aeruginosa p-hydroxybenzoate hydroxylase (PaPobA), the catalytic reactions of extremely diverse putative PHBH isozymes remain unresolved. We analyzed the phylogenetic relationships of known and predicted PHBHs and identified eight divergent clades. Clade F contains a protein that lacks the critical amino acid residues required for PaPobA to generate PHBH activity. Among proteins in this clade, Xylophilus ampelinus PobA (XaPobA) preferred PCA as a substrate and is the first known natural PCA 5-hydroxylase (PCAH). Crystal structures and kinetic properties revealed similar mechanisms of substrate carboxy group recognition between XaPobA and PaPobA. The unique Ile75, Met72, Val199, Trp201, and Phe385 residues of XaPobA form the bottom of a hydrophobic cavity with a shape that complements the 3-and 4-hydroxy groups of PCA and its binding site configuration. An interaction between the δ-sulfur atom of Met210 and the aromatic ring of PCA is likely to stabilize XaPobA-PCA complexes. The 4-hydroxy group of PCA forms a hydrogen bond with the main chain carbonyl of Thr294. These modes of binding constitute a novel substrate recognition mechanism that PaPobA lacks. This mechanism characterizes XaPobA and sheds light on the diversity of catalytic mechanisms of PobA-type PHBHs and group A flavoprotein monooxygenases.

Clinical and genetic features of patients suffering from CMT4J.

Mutations in the FIG4 gene have been identified in various diseases, including amyotrophic lateral sclerosis, Parkinson's disease, and Charcot-Marie-Tooth 4 J (CMT4J), with a wide range of phenotypic manifestations. We present eight cases of CMT4J patients carrying the p.Ile41Thr mutation of FIG4. The patients were categorized according to their phenotype. Six patients had a pure CMT; whereas, two patients had a CMT associated with parkinsonism. Three patients had an early onset and exhibited more severe forms of the disease. Three others experienced symptoms in their teenage years and had milder forms. Two patients had a late onset in adulthood. Four patients showed electrophysiological evidence of conduction blocks, typically associated with acquired neuropathies. Consequently, two of them received intravenous immunoglobulin treatment without a significant objective response. Interestingly, two heterozygous patients with the same mutations exhibited contrasting phenotypes, one having a severe early-onset form and the other experiencing a slow disease progression starting at the age of 49. Notably, although 7 out of 8 patients in this study were compound heterozygous for the p.Ile41Thr mutation, only one individual was found to be homozygous for this genetic variant and exhibited an early-onset, severe form of the disease. Additionally, one patient who developed the disease in his youth was also diagnosed with hereditary neuropathy with pressure palsies. Our findings provide insights into the CMT4J subtype by reporting on eight heterogeneous patient cases and highlight the potential for misdiagnosis when conduction blocks or asymmetrical nerve conduction study results are observed in patients with FIG4 mutations.

The IbeA protein from adherent invasive Escherichia coli is a flavoprotein sharing structural homology with FAD-dependent oxidoreductases.

Invasion of brain endothelium protein A (IbeA) is a virulence factor specific to pathogenic Escherichia coli. Originally identified in the K1 strain causing neonatal meningitis, it was more recently found in avian pathogenic Escherichia coli (APEC) and adherent invasive Escherichia coli (AIEC). In these bacteria, IbeA facilitates host cell invasion and intracellular survival, in particular, under harsh conditions like oxidative stress. Furthermore, IbeA from AIEC contributes to intramacrophage survival and replication, thus enhancing the inflammatory response within the intestine. Therefore, this factor is a promising drug target for anti-AIEC strategies in the context of Crohn's disease. Despite such an important role, the biological function of IbeA remains largely unknown. In particular, its exact nature and cellular localization, i.e., membrane-bound invasin versus cytosolic factor, are still of debate. Here, we developed an efficient protocol for recombinant expression of IbeA under native conditions and demonstrated that IbeA from AIEC is a soluble, homodimeric flavoprotein. Using mass spectrometry and tryptophan fluorescence measurements, we further showed that IbeA preferentially binds flavin adenine dinucleotide (FAD), with an affinity in the one-hundred nanomolar range and optimal binding under reducing conditions. 3D-modeling with AlphaFold revealed that IbeA shares strong structural homology with FAD-dependent oxidoreductases. Finally, we used ligand docking, mutational analyses, and molecular dynamics simulations to identify the FAD binding pocket within IbeA and characterize possible conformational changes occurring upon ligand binding. Overall, we suggest that the role of IbeA in the survival of AIEC within host cells, notably macrophages, is linked to modulation of redox processes.

Fixing Flavins: Hijacking a Flavin Transferase for Equipping Flavoproteins with a Covalent Flavin Cofactor.

Most flavin-dependent enzymes contain a dissociable flavin cofactor. We present a new approach for installing in vivo a covalent bond between a flavin cofactor and its host protein. By using a flavin transferase and carving a flavinylation motif in target proteins, we demonstrate that "dissociable" flavoproteins can be turned into covalent flavoproteins. Specifically, four different flavin mononucleotide-containing proteins were engineered to undergo covalent flavinylation: a light-oxygen-voltage domain protein, a mini singlet oxygen generator, a nitroreductase, and an old yellow enzyme-type ene reductase. Optimizing the flavinylation motif and expression conditions led to the covalent flavinylation of all four flavoproteins. The engineered covalent flavoproteins retained function and often exhibited improved performance, such as higher thermostability or catalytic performance. The crystal structures of the designed covalent flavoproteins confirmed the designed threonyl-phosphate linkage. The targeted flavoproteins differ in fold and function, indicating that this method of introducing a covalent flavin-protein bond is a powerful new method to create flavoproteins that cannot lose their cofactor, boosting their performance.

Structural and Functional Analyses of the Flavoprotein Disulfide Reductase FN0820 of Fusobacterium nucleatum.

Escherichia coli RclA and Staphylococcus aureus MerA are part of the Group I flavoprotein disulfide reductase (FDR) family and have been implicated in the contribution to bacterial pathogenesis by defending against the host immune response. Fusobacterium nucleatum is a pathogenic, anaerobic Gram-negative bacterial species commonly found in the human oral cavity and gastrointestinal tract. In this study, we discovered that the F. nucleatum protein FN0820, belonging to the Group I FDR family, exhibited a higher activity of a Cu2+-dependent NADH oxidase than E. coli RclA. Moreover, FN0820 decreased the dissolved oxygen level in the solution with higher NADH oxidase activity. We found that L-tryptophan and its analog 5-hydroxytryptophan inhibit the FN0820 activities of NADH oxidase and the concomitant reduction of oxygen. Our results have implications for developing new treatment strategies against pathogens that defend the host immune response with Group I FDRs.

Further Characterization of the Neuroendocrine Phenotype Associated With the PPOX-Related Variegate Porphyria.

BACKGROUND: Variegate porphyria is caused by mutations in the PPOX gene; it usually presents in adolescents and adults as an autosomal dominant condition, with cutaneous features or acute peripheral and/or central nervous system crises. A rarer variant, homozygous variegate porphyria, presents in childhood with cutaneous manifestations as well as neurophenotypes. This study sought to further characterize the homozygous PPOX-related neuroendocrine phenotype. METHODS: This study is a retrospective review of the patients' charts, including their clinical evaluation and molecular genetics, neurodiagnostic, and neuroradiological investigations. RESULTS: We describe here three children from a consanguineous family who presented with nystagmus, developmental delay and ataxia, photosensitive skin manifestations, and adrenal insufficiency. Analysis of porphyrins in plasma, urine, and stool together with a genetic study of the PPOX gene confirmed the diagnosis. Interestingly, brain MRI showed severe hypomyelination, a finding rarely reported in variegate porphyria, together with adrenal insufficiency. CONCLUSION: We recommend analysis of porphyrins in unexplained hypomyelination disorders. Patients with variegate porphyria should be tested for adrenal insufficiency.

Efficient Oxidation of 5-Hydroxymethylfurfural Using a Flavoprotein Oxidase from the Honeybee Apis mellifera.

The chemical 5-hydroxymethylfurfural (HMF) can be derived from lignocellulose and is an interesting bio-based platform chemical as it has the potential to be transformed into numerous valuable building blocks such as the polymer-precursor 2,5-diformylfuran (DFF). To date, only a few oxidases acting on HMF are known and by sampling atypical species, we discovered a novel flavin-dependent oxidoreductase from the honeybee Apis mellifera (beeHMFO). The enzyme can perform the chemoselective oxidation of HMF to DFF but can also readily accept other aromatic alcohols as substrates. The function of the enzyme may well be the antimicrobial generation of hydrogen peroxide using HMF, which is very abundant in honey. The discovery of this insect-derived flavoprotein oxidase holds promising potential in the synthesis of renewable products and demonstrates that insects can be an interesting source of novel biocatalysts.

Assessing the Performance of Non-Equilibrium Thermodynamic Integration in Flavodoxin Redox Potential Estimation.

Flavodoxins are enzymes that contain the redox-active flavin mononucleotide (FMN) cofactor and play a crucial role in numerous biological processes, including energy conversion and electron transfer. Since the redox characteristics of flavodoxins are significantly impacted by the molecular environment of the FMN cofactor, the evaluation of the interplay between the redox properties of the flavin cofactor and its molecular surroundings in flavoproteins is a critical area of investigation for both fundamental research and technological advancements, as the electrochemical tuning of flavoproteins is necessary for optimal interaction with redox acceptor or donor molecules. In order to facilitate the rational design of biomolecular devices, it is imperative to have access to computational tools that can accurately predict the redox potential of both natural and artificial flavoproteins. In this study, we have investigated the feasibility of using non-equilibrium thermodynamic integration protocols to reliably predict the redox potential of flavodoxins. Using as a test set the wild-type flavodoxin from Clostridium Beijerinckii and eight experimentally characterized single-point mutants, we have computed their redox potential. Our results show that 75% (6 out of 8) of the calculated reaction free energies are within 1 kcal/mol of the experimental values, and none exceed an error of 2 kcal/mol, confirming that non-equilibrium thermodynamic integration is a trustworthy tool for the quantitative estimation of the redox potential of this biologically and technologically significant class of enzymes.

Reaction mechanism and kinetics of the two-component flavoprotein dimethyl sulfone monooxygenase system: Using hydrogen peroxide for monooxygenation and substrate cleavage.

The dimethyl sulfone monooxygenase system is a two-component flavoprotein, catalyzing the monooxygenation of dimethyl sulfone (DMSO2 ) by oxidative cleavage producing methanesulfinate and formaldehyde. The reductase component (DMSR) is a flavoprotein with FMN as a cofactor, catalyzing flavin reduction using NADH. The monooxygenase (DMSMO) uses reduced flavin from the reductase and oxygen for substrate monooxygenation. DMSMO can bind to FMN and FMNH- with a Kd of 17.4 ± 0.9 µm and 4.08 ± 0.8 µm, respectively. The binding of FMN to DMSMO is required prior to binding DMSO2 . This also applies to the fast binding of reduced FMN to DMSMO followed by DMSO2 . Substituting reduced DMSR with FMNH- demonstrated the same oxidation kinetics, indicating that FMNH- from DMSR was transferred to DMSMO. The oxidation of FMNH- :DMSMO, with and without DMSO2 did not generate any flavin adducts for monooxygenation. Therefore, H2 O2 is likely to be the reactive agent to attack the substrate. The H2 O2 assay results demonstrated production of H2 O2 from the oxidation of FMNH- :DMSMO, whereas H2 O2 was not detected in the presence of DMSO2 , confirming H2 O2 utilization. The rate constant for methanesulfinate formation determined from rapid quenched flow and the rate constant for flavin oxidation were similar, indicating that H2 O2 rapidly reacts with DMSO2 , with flavin oxidation as the rate-limiting step. This is the first report of the kinetic mechanisms of both components using rapid kinetics and of a method for methanesulfinate detection using LC-MS.

Association of Radical Chemistry with LanD Flavoprotein Activity for C-Terminal Macrocyclization of a Ribosomal Peptide by Formation of an Unsaturated Thioether Residue.

LanD flavoproteins catalyze oxidative decarboxylation of the C-terminal Cys residue of a peptide to produce an enethiol. This enethiol is highly reactive and can be coupled with an upstream dehydroamino acid through Michael addition to form S-[2-aminovinyl](3-methyl)cysteine, an unsaturated thioether residue known to be characteristic of an array of C-terminally macrocyclized, ribosomally synthesized and posttranslationally modified peptides (RiPPs). Based on a two-stage bioinformatics mining of posttranslational modifications (PTMs) related to C-terminal Cys processing, we report herein that LanD activity can couple with radical S-adenosylmethionine chemistry to provide a new unsaturated thioether residue, S-[2-aminovinyl]-3-carbamoylcysteine, by conjugating the resultant enethiol with Cß of the Asn residue in the C-terminal NxxC motif of a peptide for macrocyclization. This study furthers our understanding of the variety of PTMs involved in creating the structure diversity of macrocyclic RiPPs.

Vitamin B2 enables regulation of fasting glucose availability.

Flavin adenine dinucleotide (FAD) interacts with flavoproteins to mediate oxidation-reduction reactions required for cellular energy demands. Not surprisingly, mutations that alter FAD binding to flavoproteins cause rare inborn errors of metabolism (IEMs) that disrupt liver function and render fasting intolerance, hepatic steatosis, and lipodystrophy. In our study, depleting FAD pools in mice with a vitamin B2-deficient diet (B2D) caused phenotypes associated with organic acidemias and other IEMs, including reduced body weight, hypoglycemia, and fatty liver disease. Integrated discovery approaches revealed B2D tempered fasting activation of target genes for the nuclear receptor PPARα, including those required for gluconeogenesis. We also found PPARα knockdown in the liver recapitulated B2D effects on glucose excursion and fatty liver disease in mice. Finally, treatment with the PPARα agonist fenofibrate activated the integrated stress response and refilled amino acid substrates to rescue fasting glucose availability and overcome B2D phenotypes. These findings identify metabolic responses to FAD availability and nominate strategies for the management of organic acidemias and other rare IEMs.

Exome sequencing reveals IFT172 variants in patients with non-syndromic cholestatic liver disease.

BACKGROUND AND AIM: Gene defects contribute to the aetiology of intrahepatic cholestasis. We aimed to explore the outcome of whole-exome sequencing (WES) in a cohort of 51 patients with this diagnosis. PATIENTS AND METHODS: Both paediatric (n = 33) and adult (n = 18) patients with cholestatic liver disease of unknown aetiology were eligible. WES was used for reassessment of 34 patients (23 children) without diagnostic genotypes in ABCB11, ATP8B1, ABCB4 or JAG1 demonstrable by previous Sanger sequencing, and for primary assessment of additional 17 patients (10 children). Nasopharyngeal swab mRNA was analysed to address variant pathogenicity in two families. RESULTS: WES revealed biallelic variation in 3 ciliopathy genes (PKHD1, TMEM67 and IFT172) in 4 clinically unrelated index subjects (3 children and 1 adult), heterozygosity for a known variant in PPOX in one adult index subject, and homozygosity for an unreported splice-site variation in F11R in one child. Whereas phenotypes of the index patients with mutated PKHD1, TMEM67, and PPOX corresponded with those elsewhere reported, how F11R variation underlies liver disease remains unclear. Two unrelated patients harboured different novel biallelic variants in IFT172, a gene implicated in short-rib thoracic dysplasia 10 and Bardet-Biedl syndrome 20. One patient, a homozygote for IFT172 rs780205001 c.167A>C p.(Lys56Thr) born to first cousins, had liver disease, interpreted on biopsy aged 4y as glycogen storage disease, followed by adult-onset nephronophthisis at 25y. The other, a compound heterozygote for novel frameshift variant IFT172 NM_015662.3 c.2070del p.(Met690Ilefs*11) and 2 syntenic missense variants IFT172 rs776310391 c.157T>A p.(Phe53Ile) and rs746462745 c.164C>G p.(Thr55Ser), had a severe 8mo cholestatic episode in early infancy, with persisting hyperbilirubinemia and fibrosis on imaging studies at 17y. No patient had skeletal malformations. CONCLUSION: Our findings suggest association of IFT172 variants with non-syndromic cholestatic liver disease.

Integrated Computational Study of the Light-Activated Structure of the AppA BLUF Domain and Its Spectral Signatures.

We apply an integrated approach combining microsecond MD simulations and (polarizable) QM/MM calculations of NMR, FTIR, and UV-vis spectra to validate the structure of the light-activated form of the AppA photoreceptor, an example of blue light using flavin (BLUF) protein domain. The latter photoactivate through a proton-coupled electron transfer (PCET) that results in a tautomerization of a conserved glutamine residue in the active site, but this mechanism has never been spectroscopically proven for AppA, which has been always considered as an exception. Our simulations instead confirm that the spectral features observed upon AppA photoactivation are indeed directly connected to the tautomer form of glutamine as predicted by the PCET mechanism. In addition, we observe small but significant changes in the AppA structure, which are transmitted from the flavin binding pocket to the surface of the protein.