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.

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.

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.

Distinct effects on mRNA export factor GANP underlie neurological disease phenotypes and alter gene expression depending on intron content.

Defects in the mRNA export scaffold protein GANP, encoded by the MCM3AP gene, cause autosomal recessive early-onset peripheral neuropathy with or without intellectual disability. We extend here the phenotypic range associated with MCM3AP variants, by describing a severely hypotonic child and a sibling pair with a progressive encephalopathic syndrome. In addition, our analysis of skin fibroblasts from affected individuals from seven unrelated families indicates that disease variants result in depletion of GANP except when they alter critical residues in the Sac3 mRNA binding domain. GANP depletion was associated with more severe phenotypes compared with the Sac3 variants. Patient fibroblasts showed transcriptome alterations that suggested intron content-dependent regulation of gene expression. For example, all differentially expressed intronless genes were downregulated, including ATXN7L3B, which couples mRNA export to transcription activation by association with the TREX-2 and SAGA complexes. Our results provide insight into the molecular basis behind genotype-phenotype correlations in MCM3AP-associated disease and suggest mechanisms by which GANP defects might alter RNA metabolism.

Molecular characterization of a novel His333Arg variant of human protoporphyrinogen oxidase IX.

Variegate porphyria is caused by mutations in the protoporphyrinogen oxidase IX (PPOX, EC 1.3.3.4) gene, resulting in reduced overall enzymatic activity of PPOX in human tissues. Recently, we have identified the His333Arg mutation in the PPOX protein (PPOX(H333R)) as a putative founder mutation in the Moroccan Jewish population. Herein we report the molecular characterization of PPOX(H333R) in vitro and in cells. Purified recombinant PPOX(H333R) did not show any appreciable enzymatic activity in vitro, corroborating the clinical findings. Biophysical experiments and molecular modeling revealed that PPOX(H333R) is not folded properly and fails to adopt its native functional three-dimensional conformation due to steric clashes in the vicinity of the active site of the enzyme. On the other hand, PPOX(H333R) subcellular distribution, as evaluated by live-cell confocal microscopy, is unimpaired suggesting that the functional three-dimensional fold is not required for efficient transport of the polypeptide chain into mitochondria. Overall, the data presented here provide molecular underpinnings of the pathogenicity of PPOX(H333R) and might serve as a blueprint for deciphering whether a given PPOX variant represents a disease-causing mutation.

Dynamic association of flavin cofactors to regulate flavoprotein function.

Flavoproteins are key players in numerous redox pathways in cells. Flavin cofactors FMN and FAD confer the required chemical reactivity to flavoenzymes. In most cases, the interaction between the proteins and the flavins is noncovalent, yet stronger in comparison to other redox-active cofactors, such as NADH and NADPH. The association is considered static, but this view has started to change with the recent discovery of the dynamic association of flavins and flavoenzymes. Six cases from different organisms and various metabolic pathways are discussed here. The available mechanistic details span the range from rudimentary, as in the case of the ER-resident oxidoreductase Ero1, to comprehensive, as for the bacterial respiratory complex I. The same holds true in regard to the assumed functional role of the dynamic association presented here. More work is needed to clarify the structural and functional determinants of the known examples. Identification of new cases will help to appreciate the generality of the new principle of intracellular flavoenzyme regulation.

Chloroquine corrects enlarged lysosomes in FIG4 null cells and reduces neurodegeneration in Fig4 null mice.

Loss-of-function mutations of FIG4 impair the biosynthesis of PI(3,5)P2 and are responsible for rare genetic disorders including Yunis-Varón Syndrome and Charcot-Marie-Tooth Disease Type 4 J. Cultured cells deficient in FIG4 accumulate enlarged lysosomes with hyperacidic pH, due in part to impaired regulation of lysosomal ion channels and elevated intra-lysosomal osmotic pressure. We evaluated the effects of the FDA approved drug chloroquine, which is known to reduce lysosome acidity, on FIG4 deficient cell culture and on a mouse model. Chloroquine corrected the enlarged lysosomes in FIG4 null cells. In null mice, addition of chloroquine to the drinking water slowed progression of the disorder. Growth and mobility were dramatically improved during the first month of life, and spongiform degeneration of the nervous system was reduced. The median survival of Fig4 null mice was increased from 4 weeks for untreated mutants to 8 weeks with chloroquine treatment (p < 0.009). Chloroquine thus corrects the lysosomal swelling in cultured cells and ameliorates Fig4 deficiency in vivo. The improved phenotype of mice with complete loss of Fig4 suggests that chloroquine could be beneficial FIG2 in partial loss-of-function disorders such as Charcot-Marie-Tooth Type 4 J.

Different effects of maternal homocysteine concentration, MTHFR and MTRR genetic polymorphisms on the occurrence of fetal aneuploidy.

RESEARCH QUESTION: Do maternal homocysteine (Hcy) concentrations, MTHFR and MTRR genes have effects on the occurrence of fetal aneuploidy? DESIGN: A total of 619 aneuploidy mothers and 192 control mothers were recruited in this study. Differences in distributions of maternal MTHFR 677C>T, MTHFR 1298A>C and MTRR 66A>G genetic polymorphisms and maternal Hcy concentrations between aneuploidy mothers and control mothers were analysed. RESULTS: The maternal MTHFR 677C>T polymorphism was found to be a risk factor for the occurrence of many fetal non-mosaic aneuploidies studied here, including trisomies 13, 15, 16, 18, 21, 22, TRA and TS. The maternal MTHFR 1298A>C polymorphism was found to be a risk factor specifically associated with the occurrence of fetal trisomy 15 and fetal TS. The maternal MTRR 66A>G polymorphism was found to be a risk factor only specifically associated with the occurrence of fetal trisomy 21. The Hcy concentrations of mothers of trisomies 22, 21, 18, 16, 15 and TS fetuses were significantly higher than the Hcy concentrations of control mothers. CONCLUSIONS: Overall, data suggested an association between these maternal polymorphisms and the susceptibility of fetal non-mosaic trisomy and Turner syndrome. However, these three maternal polymorphisms had different associations with the susceptibility of different fetal aneuploidies, and the elevated maternal Hcy concentration appeared to be a likely risk factor for fetal Turner syndrome and fetal trisomies.

Charcot-Marie-Tooth disease type 4J with spastic quadriplegia, epilepsy and global developmental delay: a tale of three siblings.

Charcot-Marie-Tooth (CMT) disease is mainly a disease of peripheral nervous system and patients typically present with features of demyelinating neuropathy or axonal neuropathy or both. Rarely patients present with features of central nervous system involvement. Parkinsonism, aphemia and familial epilepsy syndrome have previously come up as case reports in association with CMT type 4 J.We hereby describe a family with 3 siblings affected with CMT4J with homozygous FIG4 mutation who presented with global developmental delay, epilepsy and spastic quadriparesis.

Spectroscopic evidence for direct flavin-flavin contact in a bifurcating electron transfer flavoprotein.

A remarkable charge transfer (CT) band is described in the bifurcating electron transfer flavoprotein (Bf-ETF) from Rhodopseudomonas palustris (RpaETF). RpaETF contains two FADs that play contrasting roles in electron bifurcation. The Bf-FAD accepts electrons pairwise from NADH, directs one to a lower-reduction midpoint potential (E°) carrier, and the other to the higher-E° electron transfer FAD (ET-FAD). Previous work noted that a CT band at 726 nm formed when ET-FAD was reduced and Bf-FAD was oxidized, suggesting that both flavins participate. However, existing crystal structures place them too far apart to interact directly. We present biochemical experiments addressing this conundrum and elucidating the nature of this CT species. We observed that RpaETF missing either FAD lacked the 726 nm band. Site-directed mutagenesis near either FAD produced altered yields of the CT species, supporting involvement of both flavins. The residue substitutions did not alter the absorption maximum of the signal, ruling out contributions from residue orbitals. Instead, we propose that the residue identities modulate the population of a protein conformation that brings the ET-flavin and Bf-flavin into direct contact, explaining the 726 nm band based on a CT complex of reduced ET-FAD and oxidized Bf-FAD. This is corroborated by persistence of the 726 nm species during gentle protein denaturation and simple density functional theory calculations of flavin dimers. Although such a CT complex has been demonstrated for free flavins, this is the first observation of such, to our knowledge, in an enzyme. Thus, Bf-ETFs may optimize electron transfer efficiency by enabling direct flavin-flavin contact.

Structure and function of the hypochlorous acid-induced flavoprotein RclA from Escherichia coli.

In response to microbial invasion, the animal immune system generates hypochlorous acid (HOCl) that kills microorganisms in the oxidative burst. HOCl toxicity is amplified in the phagosome through import of the copper cation (Cu2+). In Escherichia coli and Salmonella, the transcriptional regulator RclR senses HOCl stress and induces expression of the RclA, -B, and -C proteins involved in bacterial defenses against oxidative stress. However, the structures and biochemical roles of the Rcl proteins remain to be elucidated. In this study, we first examined the role of the flavoprotein disulfide reductase (FDR) RclA in the survival of Salmonella in macrophage phagosomes, finding that RclA promotes Salmonella survival in macrophage vacuoles containing sublethal HOCl levels. To clarify the molecular mechanism, we determined the crystal structure of RclA from E. coli at 2.9 Å resolution. This analysis revealed that the structure of homodimeric RclA is similar to those of typical FDRs, exhibiting two conserved cysteine residues near the flavin ring of the cofactor flavin adenine dinucleotide (FAD). Of note, we observed that Cu2+ accelerated RclA-mediated oxidation of NADH, leading to a lowering of oxygen levels in vitro Compared with the RclA WT enzyme, substitution of the conserved cysteine residues lowered the specificity to Cu2+ or substantially increased the production of superoxide anion in the absence of Cu2+ We conclude that RclA-mediated lowering of oxygen levels could contribute to the inhibition of oxidative bursts in phagosomes. Our study sheds light on the molecular basis for how bacteria can survive HOCl stress in macrophages.

Specialized Flavoprotein Promotes Sulfur Migration and Spiroaminal Formation in Aspirochlorine Biosynthesis.

Epidithiodiketopiperazines (ETPs) are a class of ecologically and medicinally important cyclodipeptides bearing a reactive transannular disulfide bridge. Aspirochlorine, an antifungal and toxic ETP isolated from Aspergillus oryzae used in sake brewing, deviates from the common ETP scaffold owing to its unusual ring-enlarged disulfide bridge linked to a spiroaminal ring system. Although this disulfide ring system is implicated in the biological activity of ETPs the biochemical basis for this derailment has remained a mystery. Here we report the discovery of a novel oxidoreductase (AclR) that represents the first-in-class enzyme catalyzing both a carbon-sulfur bond migration and spiro-ring formation, and that the acl pathway involves a cryptic acetylation as a prerequisite for the rearrangement. Genetic screening in A. oryzae identified aclR as the candidate for the complex biotransformation, and the aclR-deficient mutant provided the biosynthetic intermediate, unexpectedly harboring an acetyl group. In vitro assays showed that AclR alone promotes 1,2-sulfamyl migration, elimination of the acetoxy group, and spiroaminal formation. AclR features a thioredoxin oxidoreductase fold with a noncanonical CXXH motif that is distinct from the CXXC in the disulfide forming oxidase for the ETP biosynthesis. Crystallographic and mutational analyses of AclR revealed that the CXXH motif is crucial for catalysis, whereas the flavin-adenine dinucleotide is required as a support of the protein fold, and not as a redox cofactor. AclR proved to be a suitable bioinformatics handle to discover a number of related fungal gene clusters that potentially code for the biosynthesis of derailed ETP compounds. Our results highlight a specialized role of the thioredoxin oxidoreductase family enzyme in the ETP pathway and expand the chemical diversity of small molecules bearing an aberrant disulfide pharmacophore.

The PIKfyve complex regulates the early melanosome homeostasis required for physiological amyloid formation.

The metabolism of PI(3,5)P2 is regulated by the PIKfyve, VAC14 and FIG4 complex, mutations in which are associated with hypopigmentation in mice. These pigmentation defects indicate a key, but as yet unexplored, physiological relevance of this complex in the biogenesis of melanosomes. Here, we show that PIKfyve activity regulates formation of amyloid matrix composed of PMEL protein within the early endosomes in melanocytes, called stage I melanosomes. PIKfyve activity controls the membrane remodeling of stage I melanosomes, which regulates PMEL abundance, sorting and processing. PIKfyve activity also affects stage I melanosome kiss-and-run interactions with lysosomes, which are required for PMEL amyloidogenesis and the establishment of melanosome identity. Mechanistically, PIKfyve activity promotes both the formation of membrane tubules from stage I melanosomes and their release by modulating endosomal actin branching. Taken together, our data indicate that PIKfyve activity is a key regulator of the melanosomal import-export machinery that fine tunes the formation of functional amyloid fibrils in melanosomes and the maintenance of melanosome identity.This article has an associated First Person interview with the first author of the paper.

Components of the endocytic and recycling trafficking pathways interfere with the integrity of the Legionella-containing vacuole.

Legionella pneumophila requires the Dot/Icm translocation system to replicate in a vacuolar compartment within host cells. Strains lacking the translocated substrate SdhA form a permeable vacuole during residence in the host cell, exposing bacteria to the host cytoplasm. In primary macrophages, mutants are defective for intracellular growth, with a pyroptotic cell death response mounted due to bacterial exposure to the cytosol. To understand how SdhA maintains vacuole integrity during intracellular growth, we performed high-throughput RNAi screens against host membrane trafficking genes to identify factors that antagonise vacuole integrity in the absence of SdhA. Depletion of host proteins involved in endocytic uptake and recycling resulted in enhanced intracellular growth and lower levels of permeable vacuoles surrounding the ΔsdhA mutant. Of interest were three different Rab GTPases involved in these processes: Rab11b, Rab8b and Rab5 isoforms, that when depleted resulted in enhanced vacuole integrity surrounding the sdhA mutant. Proteins regulated by these Rabs are responsible for interfering with proper vacuole membrane maintenance, as depletion of the downstream effectors EEA1, Rab11FIP1, or VAMP3 rescued vacuole integrity and intracellular growth of the sdhA mutant. To test the model that specific vesicular components associated with these effectors could act to destabilise the replication vacuole, EEA1 and Rab11FIP1 showed increased density about the sdhA mutant vacuole compared with the wild type (WT) vacuole. Depletion of Rab5 isoforms or Rab11b reduced this aberrant redistribution. These findings are consistent with SdhA interfering with both endocytic and recycling membrane trafficking events that act to destabilise vacuole integrity during infection.

Production of active recombinant human aldehyde oxidase (AOX) in the baculovirus expression vector system (BEVS) and deployment in a pre-clinical fraction-of-control AOX compound exposure assay.

Human aldehyde oxidase (AOX) has emerged as a key enzyme activity for consideration in modern drug discovery. The enzyme catalyzes the oxidation of a wide variety of compounds, most notably azaheterocyclics that often form the building blocks of small molecule therapeutics. Failure to consider and assess AOX drug exposure early in the drug development cycle can have catastrophic consequences for novel compounds entering the clinic. AOX is a complex molybdopterin-containing iron-sulfur flavoprotein comprised of two identical 150 kDa subunits that has proven difficult to produce in recombinant form, and a commercial source of the purified human enzyme is currently unavailable. Thus, the potential exposure of novel drug development candidates to human AOX metabolism is usually assessed by using extracts of pooled human liver cytosol as a source of the enzyme. This can complicate the assignment of AOX-specific compound exposure due to its low activity and the presence of contaminating enzymes that may have overlapping substrate specificities. Herein is described a two-step process for the isolation of recombinant human AOX dimers to near homogeneity following production in the baculovirus expression vector system (BEVS). The deployment of this BEVS-produced recombinant human AOX as a substitute for human liver extracts in a fraction-of-control AOX compound-exposure screening assay is described. The ability to generate this key enzyme activity readily in a purified recombinant form provides for a more accurate and convenient approach to the assessment of new compound exposure to bona fide AOX drug metabolism.

Photoreaction Mechanisms of Flavoprotein Photoreceptors and Their Applications.

Three classes of flavoprotein photoreceptors, cryptochromes (CRYs), light-oxygen-voltage (LOV)-domain proteins, and blue light using FAD (BLUF)-domain proteins, have been identified that control various physiological processes in multiple organisms. Accordingly, signaling activities of photoreceptors have been intensively studied and the related mechanisms have been exploited in numerous optogenetic tools. Herein, we summarize the current understanding of photoactivation mechanisms of the flavoprotein photoreceptors and review their applications.