Loss of Neurological Disease HSAN-I-Associated Gene SPTLC2 Impairs CD8+ T Cell Responses to Infection by Inhibiting T Cell Metabolic Fitness.

Patients with the neurological disorder HSAN-I suffer frequent infections, attributed to a lack of pain sensation and failure to seek care for minor injuries. Whether protective CD8+ T cells are affected in HSAN-I patients remains unknown. Here, we report that HSAN-I-associated mutations in serine palmitoyltransferase subunit SPTLC2 dampened human T cell responses. Antigen stimulation and inflammation induced SPTLC2 expression, and murine T-cell-specific ablation of Sptlc2 impaired antiviral-T-cell expansion and effector function. Sptlc2 deficiency reduced sphingolipid biosynthetic flux and led to prolonged activation of the mechanistic target of rapamycin complex 1 (mTORC1), endoplasmic reticulum (ER) stress, and CD8+ T cell death. Protective CD8+ T cell responses in HSAN-I patient PBMCs and Sptlc2-deficient mice were restored by supplementing with sphingolipids and pharmacologically inhibiting ER stress-induced cell death. Therefore, SPTLC2 underpins protective immunity by translating extracellular stimuli into intracellular anabolic signals and antagonizes ER stress to promote T cell metabolic fitness.

Formation of keto-type ceramides in palmoplantar keratoderma based on biallelic KDSR mutations in patients.

Functional skin barrier requires sphingolipid homeostasis; 3-ketodihydrosphingosine reductase or KDSR is a key enzyme of sphingolipid anabolism catalyzing the reduction of 3-ketodihydrosphingosine to sphinganine. Biallelic mutations in the KDSR gene may cause erythrokeratoderma variabilis et progressive-4, later specified as PERIOPTER syndrome, emphasizing a characteristic periorifical and ptychotropic erythrokeratoderma. We report another patient with compound heterozygous mutations in KDSR, born with generalized harlequin ichthyosis, which progressed into palmoplantar keratoderma. To determine whether patient-associated KDSR mutations lead to KDSR substrate accumulation and/or unrecognized sphingolipid downstream products in stratum corneum (SC), we analyzed lipids of this and previously published patients with non-identical biallelic mutations in KDSR. In SC of both patients, we identified 'hitherto' unobserved skin ceramides with an unusual keto-type sphingoid base in lesional and non-lesional areas, which accounted for up to 10% of the measured ceramide species. Furthermore, an overall shorter mean chain length of free and bound sphingoid bases was observed-shorter mean chain length of free sphingoid bases was also observed in lesional psoriasis vulgaris SC, but not generally in lesional atopic dermatitis SC. Formation of keto-type ceramides is probably due to a bottle neck in metabolic flux through KDSR and a bypass by ceramide synthases, which highlights the importance of tight intermediate regulation during sphingolipid anabolism and reveals substrate deprivation as potential therapy.

Glucosylceramide Synthase Inhibitors Induce Ceramide Accumulation and Sensitize H3K27 Mutant Diffuse Midline Glioma to Irradiation.

H3K27M mutant (mut) diffuse midline glioma (DMG) is a lethal cancer with no effective cure. The glycosphingolipids (GSL) metabolism is altered in these tumors and could be exploited to develop new therapies. We tested the effect of the glucosylceramide synthase inhibitors (GSI) miglustat and eliglustat on cell proliferation, alone or in combination with temozolomide or ionizing radiation. Miglustat was included in the therapy protocol of two pediatric patients. The effect of H3.3K27 trimethylation on GSL composition was analyzed in ependymoma. GSI reduced the expression of the ganglioside GD2 in a concentration and time-dependent manner and increased the expression of ceramide, ceramide 1-phosphate, sphingosine, and sphingomyelin but not of sphingosine 1-phosphate. Miglustat significantly increased the efficacy of irradiation. Treatment with miglustat according to dose recommendations for patients with Niemann-Pick disease was well tolerated with manageable toxicities. One patient showed a mixed response. In ependymoma, a high concentration of GD2 was found only in the presence of the loss of H3.3K27 trimethylation. In conclusion, treatment with miglustat and, in general, targeting GSL metabolism may offer a new therapeutic opportunity and can be administered in close proximity to radiation therapy. Alterations in H3K27 could be useful to identify patients with a deregulated GSL metabolism.

TLR9-mediated dendritic cell activation uncovers mammalian ganglioside species with specific ceramide backbones that activate invariant natural killer T cells.

CD1d-restricted invariant natural killer T (iNKT) cells represent a heterogeneous population of lipid-reactive T cells that are involved in many immune responses, mediated through T-cell receptor (TCR)-dependent and/or independent activation. Although numerous microbial lipid antigens (Ags) have been identified, several lines of evidence have suggested the existence of relevant Ags of endogenous origin. However, the identification of their precise nature as well as the molecular mechanisms involved in their generation are still highly controversial and ill defined. Here, we identified two mammalian gangliosides-namely monosialoganglioside GM3 and disialoganglioside GD3-as endogenous activators for mouse iNKT cells. These glycosphingolipids are found in Toll-like receptor-stimulated dendritic cells (DC) as several species varying in their N-acyl fatty chain composition. Interestingly, their ability to activate iNKT cells is highly dependent on the ceramide backbone structure. Thus, both synthetic GM3 and GD3 comprising a d18:1-C24:1 ceramide backbone were able to activate iNKT cells in a CD1d-dependent manner. GM3 and GD3 are not directly recognized by the iNKT TCR and required the Ag presenting cell intracellular machinery to reveal their antigenicity. We propose a new concept in which iNKT cells can rapidly respond to pre-existing self-molecules after stress-induced structural changes in CD1d-expressing cells. Moreover, these gangliosides conferred partial protection in the context of bacterial infection. Thus, this report identified new biologically relevant lipid self-Ags for iNKT cells.

Interaction between Bacteria and the Immune System for Cancer Immunotherapy: The α-GalCer Alliance.

Non-conventional T cells, such as γδ T and invariant natural killer T (iNKT) cells, are emerging players in fighting cancer. Alpha-galactosylceramide (α-GalCer) is used as an exogenous ligand to activate iNKT cells. Human cells don't have a direct pathway producing α-GalCer, which, however, can be produced by bacteria. We searched the literature for bacteria strains that are able to produce α-GalCer and used available sequencing data to analyze their presence in human tumor tissues and their association with survival. The modulatory effect of antibiotics on the concentration of α-GalCer was analyzed in mice. The human gut bacteria Bacteroides fragilis, Bacteroides vulgatus, and Prevotella copri produce α-GalCer structures that are able to activate iNKT cells. In mice, α-GalCer was depleted upon treatment with vancomycin. The three species were detected in colon adenocarcinoma (COAD) and rectum adenocarcinoma tissues, and Prevotella copri was also detected in bone tumors and glioblastoma tissues. Bacteroides vulgatus in COAD tissues correlated with better survival. In conclusion, α-GalCer-producing bacteria are part of the human gut microbiome and can infiltrate tumor tissues. These results suggest a new mechanism of interaction between bacteria and immune cells: α-GalCer produced by bacteria may activate non-conventional T cells in tumor tissues, where they can exert a direct or indirect anti-tumor activity.

Blockade of Glycosphingolipid Synthesis Inhibits Cell Cycle and Spheroid Growth of Colon Cancer Cells In Vitro and Experimental Colon Cancer Incidence In Vivo.

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers in humans. At early stages CRC is treated by surgery and at advanced stages combined with chemotherapy. We examined here the potential effect of glucosylceramide synthase (GCS)-inhibition on CRC biology. GCS is the rate-limiting enzyme in the glycosphingolipid (GSL)-biosynthesis pathway and overexpressed in many human tumors. We suppressed GSL-biosynthesis using the GCS inhibitor Genz-123346 (Genz), NB-DNJ (Miglustat) or by genetic targeting of the GCS-encoding gene UDP-glucose-ceramide-glucosyltransferase- (UGCG). GCS-inhibition or GSL-depletion led to a marked arrest of the cell cycle in Lovo cells. UGCG silencing strongly also inhibited tumor spheroid growth in Lovo cells and moderately in HCT116 cells. MS/MS analysis demonstrated markedly elevated levels of sphingomyelin (SM) and phosphatidylcholine (PC) that occurred in a Genz-concentration dependent manner. Ultrastructural analysis of Genz-treated cells indicated multi-lamellar lipid storage in vesicular compartments. In mice, Genz lowered the incidence of experimentally induced colorectal tumors and in particular the growth of colorectal adenomas. These results highlight the potential for GCS-based inhibition in the treatment of CRC.

Species-specific differences in nonlysosomal glucosylceramidase GBA2 function underlie locomotor dysfunction arising from loss-of-function mutations.

The nonlysosomal glucosylceramidase ß2 (GBA2) catalyzes the hydrolysis of glucosylceramide to glucose and ceramide. Mutations in the human GBA2 gene have been associated with hereditary spastic paraplegia (HSP), autosomal-recessive cerebellar ataxia (ARCA), and the Marinesco-Sjögren-like syndrome. However, the underlying molecular mechanisms are ill-defined. Here, using biochemistry, immunohistochemistry, structural modeling, and mouse genetics, we demonstrate that all but one of the spastic gait locus #46 (SPG46)-connected mutations cause a loss of GBA2 activity. We demonstrate that GBA2 proteins form oligomeric complexes and that protein-protein interactions are perturbed by some of these mutations. To study the pathogenesis of GBA2-related HSP and ARCA in vivo, we investigated GBA2-KO mice as a mammalian model system. However, these mice exhibited a high phenotypic variance and did not fully resemble the human phenotype, suggesting that mouse and human GBA2 differ in function. Whereas some GBA2-KO mice displayed a strong locomotor defect, others displayed only mild alterations of the gait pattern and no signs of cerebellar defects. On a cellular level, inhibition of GBA2 activity in isolated cerebellar neurons dramatically affected F-actin dynamics and reduced neurite outgrowth, which has been associated with the development of neurological disorders. Our results shed light on the molecular mechanism underlying the pathogenesis of GBA2-related HSP and ARCA and reveal species-specific differences in GBA2 function in vivo.

CNS myelin protein 36K regulates oligodendrocyte differentiation through Notch.

In contrast to humans and other mammals, zebrafish can successfully regenerate and remyelinate central nervous system (CNS) axons following injury. In addition to common myelin proteins found in mammalian myelin, 36K protein is a major component of teleost fish CNS myelin. Although 36K is one of the most abundant proteins in zebrafish brain, its function remains unknown. Here we investigate the function of 36K using translation-blocking Morpholinos. Morphant larvae showed fewer dorsally migrated oligodendrocyte precursor cells as well as upregulation of Notch ligand. A gamma secretase inhibitor, which prevents activation of Notch, could rescue oligodendrocyte precursor cell numbers in 36K morphants, suggesting that 36K regulates initial myelination through inhibition of Notch signaling. Since 36K like other short chain dehydrogenases might act on lipids, we performed thin layer chromatography and mass spectrometry of lipids and found changes in lipid composition in 36K morphant larvae. Altogether, we suggest that during early development 36K regulates membrane lipid composition, thereby altering the amount of transmembrane Notch ligands and the efficiency of intramembrane gamma secretase processing of Notch and thereby influencing oligodendrocyte precursor cell differentiation and further myelination. Further studies on the role of 36K short chain dehydrogenase in oligodendrocyte precursor cell differentiation during remyelination might open up new strategies for remyelination therapies in human patients.

Gangliosides modulate insulin secretion by pancreatic beta cells under glucose stress.

In pancreatic beta cells, the entry of glucose and downstream signaling for insulin release is regulated by the glucose transporter 2 (Glut2) in rodents. Dysfunction of the insulin-signaling cascade may lead to diabetes mellitus. Gangliosides, sialic acid-containing glycosphingolipids (GSLs), have been reported to modulate the function of several membrane proteins.Murine islets express predominantly sialylated GSLs, particularly the simple gangliosides GM3 and GD3 having a potential modulatory role in Glut2 activity. Conditional, tamoxifen-inducible gene targeting in pancreatic islets has now shown that mice lacking the glucosylceramide synthase (Ugcg), which represents the rate-limiting enzyme in GSL biosynthesis, displayed impaired glucose uptake and showed reduced insulin secretion. Consequently, mice with pancreatic GSL deficiency had higher blood glucose levels than respective controls after intraperitoneal glucose application. High-fat diet feeding enhanced this effect. GSL-deficient islets did not show apoptosis or ER stress and displayed a normal ultrastructure. Their insulin content, size and number were similar as in control islets. Isolated beta cells from GM3 synthase null mice unable to synthesize GM3 and GD3 also showed lower glucose uptake than respective control cells, corroborating the results obtained from the cell-specific model. We conclude that in particular the negatively charged gangliosides GM3 and GD3 of beta cells positively influence Glut2 function to adequately respond to high glucose loads.

Identification of Acer2 as a First Susceptibility Gene for Lithium-Induced Nephrogenic Diabetes Insipidus in Mice.

BACKGROUND: Lithium, mainstay treatment for bipolar disorder, causes nephrogenic diabetes insipidus and hypercalcemia in about 20% and 10% of patients, respectively, and may lead to acidosis. These adverse effects develop in only a subset of patients treated with lithium, suggesting genetic factors play a role. METHODS: To identify susceptibility genes for lithium-induced adverse effects, we performed a genome-wide association study in mice, which develop such effects faster than humans. On day 8 and 10 after assigning female mice from 29 different inbred strains to normal chow or lithium diet (40 mmol/kg), we housed the animals for 48 hours in metabolic cages for urine collection. We also collected blood samples. RESULTS: In 17 strains, lithium treatment significantly elevated urine production, whereas the other 12 strains were not affected. Increased urine production strongly correlated with lower urine osmolality and elevated water intake. Lithium caused acidosis only in one mouse strain, whereas hypercalcemia was found in four strains. Lithium effects on blood pH or ionized calcium did not correlate with effects on urine production. Using genome-wide association analyses, we identified eight gene-containing loci, including a locus containing Acer2, which encodes a ceramidase and is specifically expressed in the collecting duct. Knockout of Acer2 led to increased susceptibility for lithium-induced diabetes insipidus development. CONCLUSIONS: We demonstrate that genome-wide association studies in mice can be used successfully to identify susceptibility genes for development of lithium-induced adverse effects. We identified Acer2 as a first susceptibility gene for lithium-induced diabetes insipidus in mice.

Bacterial immunogenic α-galactosylceramide identified in the murine large intestine: dependency on diet and inflammation.

The glycosphingolipid, α-galactosylceramide (αGalCer), when presented by CD1d on antigen-presenting cells, efficiently activates invariant natural killer T (iNKT) cells. Thereby, it modulates immune responses against tumors, microbial and viral infections, and autoimmune diseases. Recently, the production of αGalCer by Bacteroidetes from the human gut microbiome was elucidated. Using hydrophilic interaction chromatography coupled to MS2, we screened murine intestinal tracts to identify and quantify αGalCers, and we investigated the αGalCer response to different dietary and physiologic conditions. In both the cecum and the colon of mice, we found 1-15 pmol of αGalCer per milligram of protein; in contrast, mice lacking microbiota (germ-free mice) and fed identical diet did not harbor αGalCer. The identified αGalCer contained a ß(R)-hydroxylated hexadecanoyl chain N-linked to C18-sphinganine, which differed from what has been reported with Bacteroides fragilis Unlike ß-anomeric structures, but similar to αGalCers from B. fragilis, the synthetic form of the murine αGalCer induced iNKT cell activation in vitro. Last, we observed a decrease in αGalCer production in mice exposed to conditions that alter the composition of the gut microbiota, including Western type diet, colitis, and influenza A virus infection. Collectively, this study suggests that αGalCer is produced by commensals in the mouse intestine and reveals that stressful conditions causing dysbiosis alter its synthesis. The consequences of this altered production on iNKT cell-mediated local and systemic immune responses are worthy of future studies.

Renal globotriaosylceramide facilitates tubular albumin absorption and its inhibition protects against acute kidney injury.

To elucidate the physiologic function of renal globotriaosylceramide (Gb3/CD77), which up-to-date has been associated exclusively with Shiga toxin binding, we have analyzed renal function in Gb3-deficient mice. Gb3 synthase KO (Gb3S-/-) mice displayed an increased renal albumin and low molecular weight protein excretion compared to WT. Gb3 localized at the brush border and within vesicular structures in WT proximal tubules and has now been shown to be closely associated with the receptor complex megalin/cubilin and with albumin uptake. In two clinically relevant mouse models of acute kidney injury caused by myoglobin as seen in rhabdomyolysis and the aminoglycoside gentamicin, Gb3S-/- mice showed a preserved renal function and morphology, compared to WT. Pharmacologic inhibition of glucosylceramide-based glycosphingolipids, including Gb3, in WT mice corroborated the results of genetically Gb3-deficient mice. In conclusion, our data significantly advance the current knowledge on the physiologic and pathophysiologic role of Gb3 in proximal tubules, showing an involvement in the reabsorption of filtered albumin, myoglobin and the aminoglycoside gentamicin.

PNPLA1 defects in patients with autosomal recessive congenital ichthyosis and KO mice sustain PNPLA1 irreplaceable function in epidermal omega-O-acylceramide synthesis and skin permeability barrier.

Autosomal recessive congenital ichthyosis (ARCI) is a heterogeneous group of monogenic genodermatoses that encompasses non-syndromic disorders of keratinization. The pathophysiology of ARCI has been linked to a disturbance in epidermal lipid metabolism that impaired the stratum corneum function, leading to permeability barrier defects. Functional characterization of some genes involved in ARCI contributed to the identification of molecular actors involved in epidermal lipid synthesis, transport or processing. Recently, PNPLA1 has been identified as a gene causing ARCI. While other members of PNPLA family are key elements in lipid metabolism, the function of PNPLA1 remained unclear. We identified 5 novel PNPLA1 mutations in ARCI patients, mainly localized in the putative active enzymatic domain of PNPLA1. To investigate Pnpla1 biological role, we analysed Pnpla1-deficient mice. KO mice died soon after birth from severe epidermal permeability defects. Pnpla1-deficient skin presented an important impairment in the composition and organization of the epidermal lipids. Quantification of epidermal ceramide species highlighted a blockade in the production of ω-O-acylceramides with a concomitant accumulation of their precursors in the KO. The virtually loss of ω-O-acylceramides in the stratum corneum was linked to a defective lipid coverage of the resistant pericellular shell encapsulating corneocytes, the so-called cornified envelope, and most probably disorganized the extracellular lipid matrix. Finally, these defects in ω-O-acylceramides synthesis and cornified envelope formation were also evidenced in the stratum corneum from PNPLA1-mutated patients. Overall, our data support that PNPLA1/Pnpla1 is a key player in the formation of ω-O-acylceramide, a crucial process for the epidermal permeability barrier function.

Diastereomer-specific quantification of bioactive hexosylceramides from bacteria and mammals.

Mammals synthesize, cell-type specifically, the diastereomeric hexosylceramides, ß-galactosylceramide (GalCer) and ß-glucosylceramide (GlcCer), which are involved in several diseases, such as sphingolipidosis, diabetes, chronic kidney diseases, or cancer. In contrast, Bacteroides fragilis, a member of the human gut microbiome, and the marine sponge, Agelas mauritianus, produce α-GalCer, one of the most potent stimulators for invariant natural killer T cells. To dissect the contribution of these individual stereoisomers to pathologies, we established a novel hydrophilic interaction chromatography-based LC-MS2 method and separated (R > 1.5) corresponding diastereomers from each other, independent of their lipid anchors. Testing various bacterial and mammalian samples, we could separate, identify (including the lipid anchor composition), and quantify endogenous ß-GlcCer, ß-GalCer, and α-GalCer isomers without additional derivatization steps. Thereby, we show a selective decrease of ß-GlcCers versus ß-GalCers in cell-specific models of GlcCer synthase-deficiency and an increase of specific ß-GlcCers due to loss of ß-glucoceramidase 2 activity. Vice versa, ß-GalCer increased specifically when cerebroside sulfotransferase (Gal3st1) was deleted. We further confirm ß-GalCer as substrate of globotriaosylceramide synthase for galabiaosylceramide synthesis and identify additional members of the human gut microbiome to contain immunogenic α-GalCers. Finally, this method is shown to separate corresponding hexosylsphingosine standards, promoting its applicability in further investigations.

Male meiotic cytokinesis requires ceramide synthase 3-dependent sphingolipids with unique membrane anchors.

Somatic cell cytokinesis was shown to involve the insertion of sphingolipids (SLs) to midbodies prior to abscission. Spermatogenic midbodies transform into stable intercellular bridges (ICBs) connecting clonal daughter cells in a syncytium. This process requires specialized SL structures. (1) Using high resolution-mass spectrometric imaging, we show in situ a biphasic pattern of SL synthesis with testis-specific anchors. This pattern correlates with and depends on ceramide synthase 3 (CerS3) localization in both, pachytene spermatocytes until completion of meiosis and elongating spermatids. (2) Blocking the pathways to germ cell-specific ceramides (CerS3-KO) and further to glycosphingolipids (glucosylceramide synthase-KO) in mice highlights the need for special SLs for spermatid ICB stability. In contrast to somatic mitosis these SLs require ultra-long polyunsaturated anchors with unique physico-chemical properties, which can only be provided by CerS3. Loss of these anchors causes enhanced apoptosis during meiosis, formation of multinuclear giant cells and spermatogenic arrest. Hence, testis-specific SLs, which we also link to CerS3 in human testis, are quintessential for male fertility.

Zeb1 affects epithelial cell adhesion by diverting glycosphingolipid metabolism.

This study proposes that the transcription factor Zeb1 modulates epithelial cell adhesion by diverting glycosphingolipid metabolism. Zeb1 promotes expression of a-series glycosphingolipids via regulating expression of GM3 synthase (St3gal5), which mechanistically involves Zeb1 binding to the St3gal5 promoter as well as suppressing microRNA-mediated repression of St3gal5. Functionally, the repression of St3gal5 suffices to elevate intercellular adhesion and expression of distinct junction-associated proteins, reminiscent of knockdown of Zeb1. Conversely, overexpressing St3gal5 sensitizes cells towards TGF-ß1-induced disruption of cell-cell interaction and partially antagonizes elevation of intercellular adhesion imposed by Zeb1 knockdown. These results highlight a direct connection of glycosphingolipid metabolism and epithelial cell adhesion via Zeb1.

The Crystal Structure of RosB: Insights into the Reaction Mechanism of the First Member of a Family of Flavodoxin-like Enzymes.

8-demethyl-8-aminoriboflavin-5'-phosphate (AFP) synthase (RosB) catalyzes the key reaction of roseoflavin biosynthesis by forming AFP from riboflavin-5'-phosphate (RP) and glutamate via the intermediates 8-demethyl-8-formylriboflavin-5'-phosphate (OHC-RP) and 8-demethyl-8-carboxylriboflavin-5'-phosphate (HO2 C-RP). To understand this reaction in which a methyl substituent of an aromatic ring is replaced by an amine we structurally characterized RosB in complex with OHC-RP (2.0 Å) and AFP (1.7 Å). RosB is composed of four flavodoxin-like subunits which have been upgraded with specific extensions and a unique C-terminal arm. It appears that RosB has evolved from an electron- or hydride-transferring flavoprotein to a sophisticated multi-step enzyme which uses RP as a substrate (and not as a cofactor). Structure-based active site analysis was complemented by mutational and isotope-based mass-spectrometric data to propose an enzymatic mechanism on an atomic basis.

Sulfatides are required for renal adaptation to chronic metabolic acidosis.

Urinary ammonium excretion by the kidney is essential for renal excretion of sufficient amounts of protons and to maintain stable blood pH. Ammonium secretion by the collecting duct epithelia accounts for the majority of urinary ammonium; it is driven by an interstitium-to-lumen NH3 gradient due to the accumulation of ammonium in the medullary and papillary interstitium. Here, we demonstrate that sulfatides, highly charged anionic glycosphingolipids, are important for maintaining high papillary ammonium concentration and increased urinary acid elimination during metabolic acidosis. We disrupted sulfatide synthesis by a genetic approach along the entire renal tubule. Renal sulfatide-deficient mice had lower urinary pH accompanied by lower ammonium excretion. Upon acid diet, they showed impaired ammonuria, decreased ammonium accumulation in the papilla, and chronic hyperchloremic metabolic acidosis. Expression levels of ammoniagenic enzymes and Na(+)-K(+)/NH4(+)-2Cl(-) cotransporter 2 were higher, and transepithelial NH3 transport, examined by in vitro microperfusion of cortical and outer medullary collecting ducts, was unaffected in mutant mice. We therefore suggest that sulfatides act as counterions for interstitial ammonium facilitating its retention in the papilla. This study points to a seminal role of sulfatides in renal ammonium handling, urinary acidification, and acid-base homeostasis.

Identification of the Key Enzyme of Roseoflavin Biosynthesis.

The bacteria Streptomyces davawensis and Streptomyces cinnabarinus produce roseoflavin, the only known natural riboflavin (vitamin B2 ) analogue with antibiotic activity. Roseoflavin can be considered a natural antimetabolite and has been postulated to be biosynthesized from riboflavin via the key intermediate 8-demethyl-8-aminoriboflavin (AF). The required site-specific substitution of one of the methyl groups on the dimethylbenzene ring of riboflavin by an amino group (to give AF) is challenging. The pathway from riboflavin to AF has remained elusive, and the corresponding enzyme/s was/were unknown. Herein, we show by systematic gene deletion, heterologous gene expression, and biochemical studies that the enzyme specified by the gene BN159_7989 from S. davawensis is able to carry out a whole set of chemical reactions starting from riboflavin-5'-phosphate to give the final product 8-demethyl-8-aminoriboflavin-5'-phosphate (AFP).