Insights into Lysosomal PI(3,5)P2 Homeostasis from a Structural-Biochemical Analysis of the PIKfyve Lipid Kinase Complex.

The phosphoinositide PI(3,5)P2, generated exclusively by the PIKfyve lipid kinase complex, is key for lysosomal biology. Here, we explore how PI(3,5)P2 levels within cells are regulated. We find the PIKfyve complex comprises five copies of the scaffolding protein Vac14 and one copy each of the lipid kinase PIKfyve, generating PI(3,5)P2 from PI3P and the lipid phosphatase Fig4, reversing the reaction. Fig4 is active as a lipid phosphatase in the ternary complex, whereas PIKfyve within the complex cannot access membrane-incorporated phosphoinositides due to steric constraints. We find further that the phosphoinositide-directed activities of both PIKfyve and Fig4 are regulated by protein-directed activities within the complex. PIKfyve autophosphorylation represses its lipid kinase activity and stimulates Fig4 lipid phosphatase activity. Further, Fig4 is also a protein phosphatase acting on PIKfyve to stimulate its lipid kinase activity, explaining why catalytically active Fig4 is required for maximal PI(3,5)P2 production by PIKfyve in vivo.

The chloride antiporter CLCN7 is a modifier of lysosome dysfunction in FIG4 and VAC14 mutants.

The phosphatase FIG4 and the scaffold protein VAC14 function in the biosynthesis of PI(3,5)P2, a signaling lipid that inhibits the lysosomal chloride transporter ClC-7. Loss-of-function mutations of FIG4 and VAC14 reduce PI(3,5)P2 and result in lysosomal disorders characterized by accumulation of enlarged lysosomes and neurodegeneration. Similarly, a gain of function mutation of CLCN7 encoding ClC-7 also results in enlarged lysosomes. We therefore tested the ability of reduced CLCN7 expression to compensate for loss of FIG4 or VAC14. Knock-out of CLCN7 corrected lysosomal swelling and partially corrected lysosomal hyperacidification in FIG4 null cell cultures. Knockout of the related transporter CLCN6 (ClC-6) in FIG4 null cells did not affect the lysosome phenotype. In the Fig4 null mouse, reduction of ClC-7 by expression of the dominant negative CLCN7 variant p.Gly215Arg improved growth and neurological function and increased lifespan by 20%. These observations demonstrate a role for the CLCN7 chloride transporter in pathogenesis of FIG4 and VAC14 disorders. Reduction of CLCN7 provides a new target for treatment of FIG4 and VAC14 deficiencies that lack specific therapies, such as Charcot-Marie-Tooth Type 4J and Yunis-Varón syndrome.

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.

Ultrafast photooxidation of protein-bound anionic flavin radicals.

The photophysical properties of anionic semireduced flavin radicals are largely unknown despite their importance in numerous biochemical reactions. Here, we studied the photoproducts of these intrinsically unstable species in five different flavoprotein oxidases where they can be stabilized, including the well-characterized glucose oxidase. Using ultrafast absorption and fluorescence spectroscopy, we unexpectedly found that photoexcitation systematically results in the oxidation of protein-bound anionic flavin radicals on a time scale of less than ∼100 fs. The thus generated photoproducts decay back in the remarkably narrow 10- to 20-ps time range. Based on molecular dynamics and quantum mechanics computations, positively charged active-site histidine and arginine residues are proposed to be the electron acceptor candidates. Altogether, we established that, in addition to the commonly known and extensively studied photoreduction of oxidized flavins in flavoproteins, the reverse process (i.e., the photooxidation of anionic flavin radicals) can also occur. We propose that this process may constitute an excited-state deactivation pathway for protein-bound anionic flavin radicals in general. This hitherto undocumented photochemical reaction in flavoproteins further extends the family of flavin photocycles.

Fine spectral tuning of a flavin-binding fluorescent protein for multicolor imaging.

Flavin-binding fluorescent proteins are promising genetically encoded tags for microscopy. However, spectral properties of their chromophores (riboflavin, flavin mononucleotide, and flavin adenine dinucleotide) are notoriously similar even between different protein families, which limits applications of flavoproteins in multicolor imaging. Here, we present a palette of 22 finely tuned fluorescent tags based on the thermostable LOV domain from Chloroflexus aggregans. We performed site saturation mutagenesis of three amino acid positions in the flavin-binding pocket, including the photoactive cysteine, to obtain variants with fluorescence emission maxima uniformly covering the wavelength range from 486 to 512 nm. We demonstrate three-color imaging based on spectral separation and two-color fluorescence lifetime imaging of bacteria, as well as two-color imaging of mammalian cells (HEK293T), using the proteins from the palette. These results highlight the possibility of fine spectral tuning of flavoproteins and pave the way for further applications of flavin-binding fluorescent proteins in fluorescence microscopy.

Noncovalent interactions that tune the reactivities of the flavins in bifurcating electron transferring flavoprotein.

Bifurcating electron transferring flavoproteins (Bf-ETFs) tune chemically identical flavins to two contrasting roles. To understand how, we used hybrid quantum mechanical molecular mechanical calculations to characterize noncovalent interactions applied to each flavin by the protein. Our computations replicated the differences between the reactivities of the flavins: the electron transferring flavin (ETflavin) was calculated to stabilize anionic semiquinone (ASQ) as needed to execute its single-electron transfers, whereas the Bf flavin (Bfflavin) was found to disfavor the ASQ state more than does free flavin and to be less susceptible to reduction. The stability of ETflavin ASQ was attributed in part to H-bond donation to the flavin O2 from a nearby His side chain, via comparison of models employing different tautomers of His. This H-bond between O2 and the ET site was uniquely strong in the ASQ state, whereas reduction of ETflavin to the anionic hydroquinone (AHQ) was associated with side chain reorientation, backbone displacement, and reorganization of its H-bond network including a Tyr from the other domain and subunit of the ETF. The Bf site was less responsive overall, but formation of the Bfflavin AHQ allowed a nearby Arg side chain to adopt an alternative rotamer that can H-bond to the Bfflavin O4. This would stabilize the anionic Bfflavin and rationalize effects of mutation at this position. Thus, our computations provide insights on states and conformations that have not been possible to characterize experimentally, offering explanations for observed residue conservation and raising possibilities that can now be tested.

The Pseudomonas aeruginosa PAO1 metallo flavoprotein d-2-hydroxyglutarate dehydrogenase requires Zn2+ for substrate orientation and activation.

Pseudomonas aeruginosa PAO1 d-2-hydroxyglutarate (D2HG) dehydrogenase (PaD2HGDH) oxidizes D2HG to 2-ketoglutarate during the vital l-serine biosynthesis and is a potential therapeutic target against P. aeruginosa. PaD2HGDH, which oxidizes d-malate as an alternative substrate, has been demonstrated to be a metallo flavoprotein that requires Zn2+ for activity. However, the role of Zn2+ in the enzyme has not been elucidated, making it difficult to rationalize why nature employs both a redox center and a metal ion for catalysis in PaD2HGDH and other metallo flavoenzymes. In this study, recombinant His-tagged PaD2HGDH was purified to high levels in the presence of Zn2+ or Co2+ to investigate the metal's role in catalysis. We found that the flavin reduction step was reversible and partially rate limiting for the enzyme's turnover at pH 7.4 with either D2HG or d-malate with similar rate constants for both substrates, irrespective of whether Zn2+ or Co2+ was bound to the enzyme. The steady-state pL profiles of the kcat and kcat/Km values with d-malate demonstrate that Zn2+ mediates the activation of water coordinated to the metal. Our data are consistent with a dual role for the metal, which orients the hydroxy acid substrate in the enzyme's active site and rapidly deprotonates the substrate to yield an alkoxide species for hydride transfer to the flavin. Thus, we propose a catalytic mechanism for PaD2HGDH oxidation that establishes Zn2+ as a cofactor required for substrate orientation and activation during enzymatic turnover.

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.

Coordinated missplicing of TMEM14C and ABCB7 causes ring sideroblast formation in SF3B1-mutant myelodysplastic syndrome.

SF3B1 splicing factor mutations are near-universally found in myelodysplastic syndromes (MDS) with ring sideroblasts (RS), a clonal hematopoietic disorder characterized by abnormal erythroid cells with iron-loaded mitochondria. Despite this remarkably strong genotype-to-phenotype correlation, the mechanism by which mutant SF3B1 dysregulates iron metabolism to cause RS remains unclear due to an absence of physiological models of RS formation. Here, we report an induced pluripotent stem cell model of SF3B1-mutant MDS that for the first time recapitulates robust RS formation during in vitro erythroid differentiation. Mutant SF3B1 induces missplicing of ∼100 genes throughout erythroid differentiation, including proposed RS driver genes TMEM14C, PPOX, and ABCB7. All 3 missplicing events reduce protein expression, notably occurring via 5' UTR alteration, and reduced translation efficiency for TMEM14C. Functional rescue of TMEM14C and ABCB7, but not the non-rate-limiting enzyme PPOX, markedly decreased RS, and their combined rescue nearly abolished RS formation. Our study demonstrates that coordinated missplicing of mitochondrial transporters TMEM14C and ABCB7 by mutant SF3B1 sequesters iron in mitochondria, causing RS formation.

Architecture of the mycobacterial succinate dehydrogenase with a membrane-embedded Rieske FeS cluster.

Complex II, also known as succinate dehydrogenase (SQR) or fumarate reductase (QFR), is an enzyme involved in both the Krebs cycle and oxidative phosphorylation. Mycobacterial Sdh1 has recently been identified as a new class of respiratory complex II (type F) but with an unknown electron transfer mechanism. Here, using cryoelectron microscopy, we have determined the structure of Mycobacterium smegmatis Sdh1 in the presence and absence of the substrate, ubiquinone-1, at 2.53-Å and 2.88-Å resolution, respectively. Sdh1 comprises three subunits, two that are water soluble, SdhA and SdhB, and one that is membrane spanning, SdhC. Within these subunits we identified a quinone-binding site and a rarely observed Rieske-type [2Fe-2S] cluster, the latter being embedded in the transmembrane region. A mutant, where two His ligands of the Rieske-type [2Fe-2S] were changed to alanine, abolished the quinone reduction activity of the Sdh1. Our structures allow the proposal of an electron transfer pathway that connects the substrate-binding and quinone-binding sites. Given the unique features of Sdh1 and its essential role in Mycobacteria, these structures will facilitate antituberculosis drug discovery efforts that specifically target this complex.

Cryoelectron microscopy structure and mechanism of the membrane-associated electron-bifurcating flavoprotein Fix/EtfABCX.

The electron-transferring flavoprotein-menaquinone oxidoreductase ABCX (EtfABCX), also known as FixABCX for its role in nitrogen-fixing organisms, is a member of a family of electron-transferring flavoproteins that catalyze electron bifurcation. EtfABCX enables endergonic reduction of ferredoxin (E°' ∼-450 mV) using NADH (E°' -320 mV) as the electron donor by coupling this reaction to the exergonic reduction of menaquinone (E°' -80 mV). Here we report the 2.9 Å structure of EtfABCX, a membrane-associated flavin-based electron bifurcation (FBEB) complex, from a thermophilic bacterium. EtfABCX forms a superdimer with two membrane-associated EtfCs at the dimer interface that contain two bound menaquinones. The structure reveals that, in contrast to previous predictions, the low-potential electrons bifurcated from EtfAB are most likely directly transferred to ferredoxin, while high-potential electrons reduce the quinone via two [4Fe-4S] clusters in EtfX. Surprisingly, EtfX shares remarkable structural similarity with mammalian [4Fe-4S] cluster-containing ETF ubiquinone oxidoreductase (ETF-QO), suggesting an unexpected evolutionary link between bifurcating and nonbifurcating systems. Based on this structure and spectroscopic studies of a closely related EtfABCX, we propose a detailed mechanism of the catalytic cycle and the accompanying structural changes in this membrane-associated FBEB system.

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.

How an assembly factor enhances covalent FAD attachment to the flavoprotein subunit of complex II.

The membrane-bound complex II family of proteins is composed of enzymes that catalyze succinate and fumarate interconversion coupled with reduction or oxidation of quinones within the membrane domain. The majority of complex II enzymes are protein heterotetramers with the different subunits harboring a variety of redox centers. These redox centers are used to transfer electrons between the site of succinate-fumarate oxidation/reduction and the membrane domain harboring the quinone. A covalently bound FAD cofactor is present in the flavoprotein subunit, and the covalent flavin linkage is absolutely required to enable the enzyme to oxidize succinate. Assembly of the covalent flavin linkage in eukaryotic cells and many bacteria requires additional protein assembly factors. Here, we provide mechanistic details for how the assembly factors work to enhance covalent flavinylation. Both prokaryotic SdhE and mammalian SDHAF2 enhance FAD binding to their respective apoprotein of complex II. These assembly factors also increase the affinity for dicarboxylates to the apoprotein-noncovalent FAD complex and stabilize the preassembly complex. These findings are corroborated by previous investigations of the roles of SdhE in enhancing covalent flavinylation in both bacterial succinate dehydrogenase and fumarate reductase flavoprotein subunits and of SDHAF2 in performing the same function for the human mitochondrial succinate dehydrogenase flavoprotein. In conclusion, we provide further insight into assembly factor involvement in building complex II flavoprotein subunit active site required for succinate oxidation.

A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency.

Soluble pyridine nucleotide transhydrogenases (STHs) are flavoenzymes involved in the redox homeostasis of the essential cofactors NAD(H) and NADP(H). They catalyze the reversible transfer of reducing equivalents between the two nicotinamide cofactors. The soluble transhydrogenase from Escherichia coli (SthA) has found wide use in both in vivo and in vitro applications to steer reducing equivalents toward NADPH-requiring reactions. However, mechanistic insight into SthA function is still lacking. In this work, we present a biochemical characterization of SthA, focusing for the first time on the reactivity of the flavoenzyme with molecular oxygen. We report on oxidase activity of SthA that takes place both during transhydrogenation and in the absence of an oxidized nicotinamide cofactor as an electron acceptor. We find that this reaction produces the reactive oxygen species hydrogen peroxide and superoxide anion. Furthermore, we explore the evolutionary significance of the well-conserved CXXXXT motif that distinguishes STHs from the related family of flavoprotein disulfide reductases in which a CXXXXC motif is conserved. Our mutational analysis revealed the cysteine and threonine combination in SthA leads to better coupling efficiency of transhydrogenation and reduced reactive oxygen species release compared to enzyme variants with mutated motifs. These results expand our mechanistic understanding of SthA by highlighting reactivity with molecular oxygen and the importance of the evolutionarily conserved sequence motif.

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.

A dimer-monomer transition captured by the crystal structures of cyanobacterial apo flavodoxin.

In cyanobacteria and algae (but not plants), flavodoxin (Fld) replaces ferredoxin (Fd) under stress conditions to transfer electrons from photosystem I (PSI) to ferredoxin-NADP+ reductase (FNR) during photosynthesis. Fld constitutes a small electron carrier noncovalently bound to flavin mononucleotide (FMN), and also an ideal model for revealing the protein/flavin-binding mechanism because of its relative simplicity compared to other flavoproteins. Here, we report two crystal structures of apo-Fld from Synechococcus sp. PCC 7942, one dimeric structure of 2.09 Å and one monomeric structure of 1.84 Å resolution. Analytical ultracentrifugation showed that in solution, apo-Fld exists both as monomers and dimers. Our dimer structure contains two ligand-binding pockets separated by a distance of 45 Å, much longer than the previous structures of FMN-bound dimers. These results suggested a potential dimer-monomer transition mechanism of cyanobacterial apo-Fld. We further propose that the dimer represents the "standby" state to stabilize itself, while the monomer constitutes the "ready" state to bind FMN. Furthermore, we generated a new docking model of cyanobacterial Fld-FNR complex based on the recently reported cryo-EM structures, and mapped the special interactions between Fld and FNR in detail.

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.

Myelodysplastic syndromes with ring sideroblasts.

Myelodysplastic syndromes (MDS) are acquired bone marrow malignant disorders characterized by ineffective hematopoiesis, resulting from a complex interaction between genetic and epigenetic mutations, alterations of the marrow microenvironment, and the immune system. In 2001, the World Health Organization (WHO) proposed a classification that integrates morphologic and genetic information, considering the MDS with ring sideroblasts (MDS-RS) as a distinct entity. Considering the strong association between MDS-RS and SF3B1 mutation and its importance in the development of MDS, the last WHO classification replaced the prior entity of MDS-RS with MDS with SF3B1 mutation. Several studies were performed to explore this genotype-phenotype correlation. Mutant SF3B1 protein deregulates the expression of genes implicated in developing hematopoietic stem and progenitor cells. Of paramount importance are PPOX and ABCB7 involved in iron metabolism. Another essential role in hemopoiesis is played by the transforming growth factor-beta (TGF-ß) receptor. This gene exerts its effects on SMAD pathways, regulating hematopoiesis through effects on balancing proliferation and apoptosis cell inactivity, differentiation, and migration. Luspatercept (ACE-536) is a soluble fusion protein that inhibits molecules in the TGF-ß superfamily. Since its structure resembles the TGF-ß family receptor, it catches TGF-ß superfamily ligands before binding to the receptor, resulting in reduced activation of SMAD signaling, thus enabling erythroid maturation. Luspatercept was investigated in the phase III trial MEDALIST, showing promising efficacy in treating anemia compared to placebo. Nowadays, further studies are needed to explore the real potential of luspatercept, investigating the biological features likely associated with treatment response, the potential use in combination treatments, and its role in the treatment of naïve MDS.

T2 olivary nuclei hyperintensities: A characteristic neuroimaging finding in FIG4-related leukoencephalopathy.

FIG4 related leukoencephalopathy has recently been considered as an expanded spectrum of FIG4 related disorders characterized by upper and lower motor neuron involvement, dystonia, intellectual disability, bulbar symptoms with cerebellar atrophy. We report a 7-year-old girl who presented with classic clinical features of FIG4 related leukoencephalopathy and neuroimaging showed characteristic T2 olivary nuclei hyperintensities in addition to bilateral parietal lobe and thalamic hyperintensities and mild cerebellar atrophy. Trio exome sequencing with Sanger confirmation revealed a novel variant c.504C>G in the FIG4 gene. Phase contrast microscopy of skin fibroblast cultures detect enlarged vacuoles in 50% of patient's fibroblasts as opposed to 18.6% vacuolation in cultured control fibroblasts (p < 0.00001), a feature characteristic of fibroblasts with deleterious variants of FIG4. In addition, we have reviewed and compared the phenotypic features of published cases of FIG4 related leukoencephalopathy from literature. This case adds to the delineation of FIG4 related leukoencephalopathy phenotype. The radiological finding of T2 inferior olivary nuclei hyperintensities appear to be characteristic for the phenotype or at least for the cases due to variants in and around the 168th codon and active effort should be made to detect the same as it can add to the genotype phenotype spectrum.

Involvement of a flavoprotein, acetohydroxyacid synthase, in growth and riboflavin production in riboflavin-overproducing Ashbya gossypii mutant.

BACKGROUND: Previously, we isolated a riboflavin-overproducing Ashbya gossypii mutant (MT strain) and discovered some mutations in genes encoding flavoproteins. Here, we analyzed the riboflavin production in the MT strain, in view of flavoproteins, which are localized in the mitochondria. RESULTS: In the MT strain, mitochondrial membrane potential was decreased compared with that in the wild type (WT) strain, resulting in increased reactive oxygen species. Additionally, diphenyleneiodonium (DPI), a universal flavoprotein inhibitor, inhibited riboflavin production in the WT and MT strains at 50 µM, indicating that some flavoproteins may be involved in riboflavin production. The specific activities of NADH and succinate dehydrogenases were significantly reduced in the MT strain, but those of glutathione reductase and acetohydroxyacid synthase were increased by 4.9- and 25-fold, respectively. By contrast, the expression of AgGLR1 gene encoding glutathione reductase was increased by 32-fold in the MT strain. However, that of AgILV2 gene encoding the catalytic subunit of acetohydroxyacid synthase was increased by only 2.1-fold. These results suggest that in the MT strain, acetohydroxyacid synthase, which catalyzes the first reaction of branched-chain amino acid biosynthesis, is vital for riboflavin production. The addition of valine, which is a feedback inhibitor of acetohydroxyacid synthase, to a minimal medium inhibited the growth of the MT strain and its riboflavin production. In addition, the addition of branched-chain amino acids enhanced the growth and riboflavin production in the MT strain. CONCLUSION: The significance of branched-chain amino acids for riboflavin production in A. gossypii is reported and this study opens a novel approach for the effective production of riboflavin in A. gossypii.