Myxococcus xanthus encapsulin cargo protein EncD is a flavin-binding protein with ferric reductase activity.

Encapsulins are protein nanocompartments that regulate cellular metabolism in several bacteria and archaea. Myxococcus xanthus encapsulins protect the bacterial cells against oxidative stress by sequestering cytosolic iron. These encapsulins are formed by the shell protein EncA and three cargo proteins: EncB, EncC, and EncD. EncB and EncC form rotationally symmetric decamers with ferroxidase centers (FOCs) that oxidize Fe+2 to Fe+3 for iron storage in mineral form. However, the structure and function of the third cargo protein, EncD, have yet to be determined. Here, we report the x-ray crystal structure of EncD in complex with flavin mononucleotide. EncD forms an α-helical hairpin arranged as an antiparallel dimer, but unlike other flavin-binding proteins, it has no ß-sheet, showing that EncD and its homologs represent a unique class of bacterial flavin-binding proteins. The cryo-EM structure of EncA-EncD encapsulins confirms that EncD binds to the interior of the EncA shell via its C-terminal targeting peptide. With only 100 amino acids, the EncD α-helical dimer forms the smallest flavin-binding domain observed to date. Unlike EncB and EncC, EncD lacks a FOC, and our biochemical results show that EncD instead is a NAD(P)H-dependent ferric reductase, indicating that the M. xanthus encapsulins act as an integrated system for iron homeostasis. Overall, this work contributes to our understanding of bacterial metabolism and could lead to the development of technologies for iron biomineralization and the production of iron-containing materials for the treatment of various diseases associated with oxidative stress.

From Ground-State to Excited-State Activation Modes: Flavin-Dependent "Ene"-Reductases Catalyzed Non-natural Radical Reactions.

Enzymes are desired catalysts for chemical synthesis, because they can be engineered to provide unparalleled levels of efficiency and selectivity. Yet, despite the astonishing array of reactions catalyzed by natural enzymes, many reactivity patterns found in small molecule catalysts have no counterpart in the living world. With a detailed understanding of the mechanisms utilized by small molecule catalysts, we can identify existing enzymes with the potential to catalyze reactions that are currently unknown in nature. Over the past eight years, our group has demonstrated that flavin-dependent "ene"-reductases (EREDs) can catalyze various radical-mediated reactions with unparalleled levels of selectivity, solving long-standing challenges in asymmetric synthesis.This Account presents our development of EREDs as general catalysts for asymmetric radical reactions. While we have developed multiple mechanisms for generating radicals within protein active sites, this account will focus on examples where flavin mononucleotide hydroquinone (FMNhq) serves as an electron transfer radical initiator. While our initial mechanistic hypotheses were rooted in electron-transfer-based radical initiation mechanisms commonly used by synthetic organic chemists, we ultimately uncovered emergent mechanisms of radical initiation that are unique to the protein active site. We will begin by covering intramolecular reactions and discussing how the protein activates the substrate for reduction by altering the redox-potential of alkyl halides and templating the charge transfer complex between the substrate and flavin-cofactor. Protein engineering has been used to modify the fundamental photophysics of these reactions, highlighting the opportunity to tune these systems further by using directed evolution. This section highlights the range of coupling partners and radical termination mechanisms available to intramolecular reactions.The next section will focus on intermolecular reactions and the role of enzyme-templated ternary charge transfer complexes among the cofactor, alkyl halide, and coupling partner in gating electron transfer to ensure that it only occurs when both substrates are bound within the protein active site. We will highlight the synthetic applications available to this activation mode, including olefin hydroalkylation, carbohydroxylation, arene functionalization, and nitronate alkylation. This section also discusses how the protein can favor mechanistic steps that are elusive in solution for the asymmetric reductive coupling of alkyl halides and nitroalkanes. We are aware of several recent EREDs-catalyzed photoenzymatic transformations from other groups. We will discuss results from these papers in the context of understanding the nuances of radical initiation with various substrates.These biocatalytic asymmetric radical reactions often complement the state-of-the-art small-molecule-catalyzed reactions, making EREDs a valuable addition to a chemist's synthetic toolbox. Moreover, the underlying principles studied with these systems are potentially operative with other cofactor-dependent proteins, opening the door to different types of enzyme-catalyzed radical reactions. We anticipate that this Account will serve as a guide and inspire broad interest in repurposing existing enzymes to access new transformations.

RibU is an essential determinant of Listeria pathogenesis that mediates acquisition of FMN and FAD during intracellular growth.

Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are essential riboflavin-derived cofactors involved in a myriad of redox reactions across all forms of life. Nevertheless, the basis of flavin acquisition strategies by riboflavin auxotrophic pathogens remains poorly defined. In this study, we examined how the facultative intracellular pathogen Listeria monocytogenes, a riboflavin auxotroph, acquires flavins during infection. A L. monocytogenes mutant lacking the putative riboflavin transporter (RibU) was completely avirulent in mice but had no detectable growth defect in nutrient-rich media. However, unlike wild type, the RibU mutant was unable to grow in defined media supplemented with FMN or FAD or to replicate in macrophages starved for riboflavin. Consistent with RibU functioning to scavenge FMN and FAD inside host cells, a mutant unable to convert riboflavin to FMN or FAD retained virulence and grew in cultured macrophages and in spleens and livers of infected mice. However, this FMN- and FAD-requiring strain was unable to grow in the gallbladder or intestines, where L. monocytogenes normally grows extracellularly, suggesting that these sites do not contain sufficient flavin cofactors to promote replication. Thus, by deleting genes required to synthesize FMN and FAD, we converted L. monocytogenes from a facultative to an obligate intracellular pathogen. Collectively, these data indicate that L. monocytogenes requires riboflavin to grow extracellularly in vivo but scavenges FMN and FAD to grow in host cells.

Mapping the Intersubunit Interdomain FMN-Heme Interactions in Neuronal Nitric Oxide Synthase by Targeted Quantitative Cross-Linking Mass Spectrometry.

Nitric oxide synthase (NOS) in mammals is a family of multidomain proteins in which interdomain electron transfer (IET) is controlled by domain-domain interactions. Calmodulin (CaM) binds to the canonical CaM-binding site in the linker region between the FMN and heme domains of NOS and allows tethered FMN domain motions, enabling an intersubunit FMN-heme IET in the output state for NO production. Our previous cross-linking mass spectrometric (XL MS) results demonstrated site-specific protein dynamics in the CaM-responsive regions of rat neuronal NOS (nNOS) reductase construct, a monomeric protein [Jiang et al., Biochemistry, 2023, 62, 2232-2237]. In this work, we have extended our combined approach of XL MS structural mapping and AlphaFold structural prediction to examine the homodimeric nNOS oxygenase/FMN (oxyFMN) construct, an established model of the NOS output state. We employed parallel reaction monitoring (PRM) based quantitative XL MS (qXL MS) to assess the CaM-induced changes in interdomain dynamics and interactions. Intersubunit cross-links were identified by mapping the cross-links onto top AlphaFold structural models, which was complemented by comparing their relative abundances in the cross-linked dimeric and monomeric bands. Furthermore, contrasting the CaM-free and CaM-bound nNOS samples shows that CaM enables the formation of the intersubunit FMN-heme docking complex and that CaM binding induces extensive, allosteric conformational changes across the NOS regions. Moreover, the observed cross-links sites specifically respond to changes in ionic strength. This indicates that interdomain salt bridges are responsible for stabilizing and orienting the output state for efficient FMN-heme IET. Taken together, our targeted qXL MS results have revealed that CaM and ionic strength modulate specific dynamic changes in the CaM/FMN/heme complexes, particularly in the context of intersubunit interdomain FMN-heme interactions.

Elucidation of the Catalytic Sequence of Dihydroorotate Dehydrogenase B from Lactoccocus lactis: Evidence for Accumulation of a Flavin Bisemiquinone State in Catalysis.

The physiological role of dihydroorotate dehydrogenase (DHOD) enzymes is to catalyze the oxidation of dihydroorotate to orotate in pyrimidine biosynthesis. DHOD enzymes are structurally diverse existing as both soluble and membrane-associated forms. The Family 1 enzymes are soluble and act either as conventional single subunit flavin-dependent dehydrogenases known as Class 1A (DHODA) or as unusual heterodimeric enzymes known as Class 1B (DHODB). DHODBs possess two active sites separated by ∼20 Å, each with a noncovalently bound flavin cofactor. NAD is thought to interact at the FAD containing site, and the pyrimidine substrate is known to bind at the FMN containing site. At the approximate center of the protein is a single Fe2S2 center that is assumed to act as a conduit, facilitating one-electron transfers between the flavins. We present anaerobic transient state analysis of a DHODB enzyme from Lactoccocus lactis. The data presented primarily report the exothermic reaction that reduces orotate to dihydroorotate. The reductive half reaction reveals rapid two-electron reduction that is followed by the accumulation of a four-electron reduced state when NADH is added in excess, suggesting that the initial two electrons acquired reside on the FMN cofactor. Concomitant with the first reduction is the accumulation of a long-wavelength absorption feature consistent with the blue form of a flavin semiquinone. Spectral deconvolution and fitting to a model that includes reversibility for the second electron transfer reveals equilibrium accumulation of a flavin bisemiquinone state that has features of both red and blue semiquinones. Single turnover reactions with limiting NADH and excess orotate reveal that the flavin bisemiquinone accumulates with reduction of the enzyme by NADH and decays with reduction of the pyrimidine substrate, establishing the bisemiquinone as a fractional state of the two-electron reduced intermediate observed.

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.

Allosteric modulation of cytochrome P450 enzymes by the NADPH cytochrome P450 reductase FMN-containing domain.

NADPH-cytochrome P450 reductase delivers electrons required by heme oxygenase, squalene monooxygenase, fatty acid desaturase, and 48 human cytochrome P450 enzymes. While conformational changes supporting reductase intramolecular electron transfer are well defined, intermolecular interactions with these targets are poorly understood, in part because of their transient association. Herein the reductase FMN domain responsible for interacting with targets was fused to the N-terminus of three drug-metabolizing and two steroidogenic cytochrome P450 enzymes to increase the probability of interaction. These artificial fusion enzymes were profiled for their ability to bind their respective substrates and inhibitors and to perform catalysis supported by cumene hydroperoxide. Comparisons with the isolated P450 enzymes revealed that even the oxidized FMN domain causes substantial and diverse effects on P450 function. The FMN domain could increase, decrease, or not affect total ligand binding and/or dissociation constants depending on both P450 enzyme and ligand. As examples, FMN domain fusion has no effect on inhibitor ketoconazole binding to CYP17A1 but substantially altered CYP21A2 binding of the same compound. FMN domain fusion to CYP21A2 resulted in differential effects dependent on whether the ligand was 17α-hydroxyprogesterone versus ketoconazole. Similar enzyme-specific effects were observed on steady-state kinetics. These observations are most consistent with FMN domain interacting with the proximal P450 surface to allosterically impact P450 ligand binding and metabolism separate from electron delivery. The variety of effects on different P450 enzymes and on the same P450 with different ligands suggests intricate and differential allosteric communication between the P450 active site and its proximal reductase-binding surface.

A new member of the flavodoxin superfamily from Fusobacterium nucleatum that functions in heme trafficking and reduction of anaerobilin.

Fusobacterium nucleatum is an opportunistic oral pathogen that is associated with various cancers. To fulfill its essential need for iron, this anaerobe will express heme uptake machinery encoded at a single genetic locus. The heme uptake operon includes HmuW, a class C radical SAM-dependent methyltransferase that degrades heme anaerobically to release Fe2+ and a linear tetrapyrrole called anaerobilin. The last gene in the operon, hmuF encodes a member of the flavodoxin superfamily of proteins. We discovered that HmuF and a paralog, FldH, bind tightly to both FMN and heme. The structure of Fe3+-heme-bound FldH (1.6 Å resolution) reveals a helical cap domain appended to the ⍺/ß core of the flavodoxin fold. The cap creates a hydrophobic binding cleft that positions the heme planar to the si-face of the FMN isoalloxazine ring. The ferric heme iron is hexacoordinated to His134 and a solvent molecule. In contrast to flavodoxins, FldH and HmuF do not stabilize the FMN semiquinone but instead cycle between the FMN oxidized and hydroquinone states. We show that heme-loaded HmuF and heme-loaded FldH traffic heme to HmuW for degradation of the protoporphyrin ring. Both FldH and HmuF then catalyze multiple reductions of anaerobilin through hydride transfer from the FMN hydroquinone. The latter activity eliminates the aromaticity of anaerobilin and the electrophilic methylene group that was installed through HmuW turnover. Hence, HmuF provides a protected path for anaerobic heme catabolism, offering F. nucleatum a competitive advantage in the colonization of anoxic sites of the human body.

Structural insights into the semiquinone form of human Cytochrome P450 reductase by DEER distance measurements between a native flavin and a spin labelled non-canonical amino acid.

The flavoprotein Cytochrome P450 reductase (CPR) is the unique electron pathway from NADPH to Cytochrome P450 (CYPs). The conformational dynamics of human CPR in solution, which involves transitions from a "locked/closed" to an "unlocked/open" state, is crucial for electron transfer. To date, however, the factors guiding these changes remain unknown. By Site-Directed Spin Labelling coupled to Electron Paramagnetic Resonance spectroscopy, we have incorporated a non-canonical amino acid onto the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) domains of soluble human CPR, and labelled it with a specific nitroxide spin probe. Taking advantage of the endogenous FMN cofactor, we successfully measured for the first time, the distance distribution by DEER between the semiquinone state FMNH• and the nitroxide. The DEER data revealed a salt concentration-dependent distance distribution, evidence of an "open" CPR conformation at high salt concentrations exceeding previous reports. We also conducted molecular dynamics simulations which unveiled a diverse ensemble of conformations for the "open" semiquinone state of the CPR at high salt concentration. This study unravels the conformational landscape of the one electron reduced state of CPR, which had never been studied before.

Flavin mononucleotide regulated photochemical isomerization and degradation of zeatin.

cis-Zeatin (cZ), a cytokinin often overlooked compared to trans-zeatin (tZ), can now be controlled in live cells and plants through a new biocompatible reaction. Using flavin photosensitizers, cZ can be isomerized to tZ or degraded, depending on the presence of a reducing reagent. This breakthrough offers a novel approach for regulating plant growth through chemical molecules.

Metabolism of FAD, FMN and riboflavin (vitamin B2) in the human parasitic blood fluke Schistosoma mansoni.

BACKGROUND: Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. The intravascular worms acquire the nutrients necessary for their survival from host blood. Since all animals are auxotrophic for riboflavin (vitamin B2), schistosomes too must import it to survive. Riboflavin is an essential component of the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD); these support key functions of dozens of flavoenzymes. METHODS: Here, using a combination of metabolomics, enzyme kinetics and in silico molecular analysis, we focus on the biochemistry of riboflavin and its metabolites in Schistosoma mansoni (Sm). RESULTS: We show that when schistosomes are incubated in murine plasma, levels of FAD decrease over time while levels of FMN increase. We show that live schistosomes cleave exogenous FAD to generate FMN and this ability is significantly blocked when expression of the surface nucleotide pyrophosphatase/phosphodiesterase ectoenzyme SmNPP5 is suppressed using RNAi. Recombinant SmNPP5 cleaves FAD with a Km of 178 ± 5.9 µM and Kcat/Km of 324,734 ± 36,347 M- 1.S- 1. The FAD-dependent enzyme IL-4I1 drives the oxidative deamination of phenylalanine to produce phenylpyruvate and H2O2. Since schistosomes are damaged by H2O2, we determined if SmNPP5 could impede H2O2 production by blocking IL-4I1 action in vitro. We found that this was not the case; covalently bound FAD on IL-4I1 appears inaccessible to SmNPP5. We also report that live schistosomes can cleave exogenous FMN to generate riboflavin and this ability is significantly impeded when expression of a second surface ectoenzyme (alkaline phosphatase, SmAP) is suppressed. Recombinant SmAP cleaves FMN with a Km of 3.82 ± 0.58 mM and Kcat/Km of 1393 ± 347 M- 1.S- 1. CONCLUSIONS: The sequential hydrolysis of FAD by tegumental ecto-enzymes SmNPP5 and SmAP can generate free vitamin B2 around the worms from where it can be conveniently imported by the recently described schistosome riboflavin transporter SmaRT. Finally, we identified in silico schistosome homologs of enzymes that are involved in intracellular vitamin B2 metabolism. These are riboflavin kinase (SmRFK) as well as FAD synthase (SmFADS); cDNAs encoding these two enzymes were cloned and sequenced. SmRFK is predicted to convert riboflavin to FMN while SmFADS could further act on FMN to regenerate FAD in order to facilitate robust vitamin B2-dependent metabolism in schistosomes.

Mediating the performance of anaerobic granular sludge by exogenous flavin supplementation: Flavin binding capacity and behavior, interspecies electron transfer, and methane production.

Although flavins are known as effective electron mediators, the binding capacity of exogenous flavins by anaerobic granular sludge (AGS) and their role in interspecies electron transfer (IET) remains unknown. In this study, AGS was mediated by using three exogenous flavins of riboflavin (RF), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). Results showed that the total amounts of flavins associated with extracellular polymeric substance (EPS) of AGS increased by 2.03-2.42 and 3.83-4.94 folds, after exposure to 50 and 200 µM of exogenous flavins, respectively. A large portion of FMN and FAD was transformed into RF by AGS. Exogenous flavin mediation also stimulated the production of EPS and cytochrome c (c-Cyts) as well as cytochrome-bound flavins. The increased abundance of these electron mediators led to a reduced electrochemical impedance of EPS and improved extracellular electron transfer capacity. The methane production of AGS after mediation with exogenous RF, FMN, and FAD increased by 19.03-31.71%, 22.86-26.04%, and 28.51-33.44%, respectively. This study sheds new light on the role of exogenous flavins in promoting the IET process of a complex microbial aggregate of AGS.

Medium-term antifungal effects of methylene blue versus flavin mononucleotide in the treatment of moderate toenail onychomycosis.

BACKGROUND: Methylene blue (MB) and flavin mononucleotide (FMN)-mediated photodynamic therapy (PDT) have demonstrated local antimicrobial effect, but no direct comparative study has been published so far for the treatment of toenail onychomycosis. OBJECTIVES: To directly compare the short and medium-term efficacy of MB versus FMN as photosensitizers in PDT for toenail onychomycosis by applying them in a 40% w/w urea cream in two different dye concentrations. METHODS: Forty toenails with distal and lateral subungual moderate onychomycosis due to dermatophyte fungi were randomised to receive 10 weekly sessions of PDT mediated by four topical formulations including MB or FMN at two different concentrations: Group I: 0.1% w/w MB; Group II: 2% w/w MB; Group III: 0.1% w/w FMN; and Group IV: 2% w/w FMN. Photographs were used for onychomycosis severity index (OSI) estimation allowing clinical assessment at any point of the study. Microscopic and microbiological evaluations were carried out at baseline, 27- and 35-week follow-ups. Side effects were recorded along with patient satisfaction. RESULTS: At week 27, mycological cure rates were 60%, 30%, 50% and 40% and complete cure rates were 0%, 20%, 10% and 20%, for Groups I, II, III and IV respectively. At week 35, mycological cure rates were 70%, 70%, 70% and 60% and complete cure rates were 30%, 50%, 70% and 30%, for Groups I, II, III and IV respectively. All cream formulations were safe and patients were fairly satisfied. CONCLUSIONS: Results of the present work confirm PDT as a therapeutic alternative for onychomycosis. Although all cream formulations were safe and effective, with a good degree of satisfaction, higher cure rates were obtained with 2% w/w MB cream and 0.1% w/w FMN cream.

Expanding the DNA-encoded library toolbox: identifying small molecules targeting RNA.

DNA-encoded library (DEL) technology is a powerful tool for small molecule identification in drug discovery, yet the reported DEL selection strategies were applied primarily on protein targets in either purified form or in cellular context. To expand the application of this technology, we employed DEL selection on an RNA target HIV-1 TAR (trans-acting responsive region), but found that the majority of signals were resulted from false positive DNA-RNA binding. We thus developed an optimized selection strategy utilizing RNA patches and competitive elution to minimize unwanted DNA binding, followed by k-mer analysis and motif search to differentiate false positive signal. This optimized strategy resulted in a very clean background in a DEL selection against Escherichia coli FMN Riboswitch, and the enriched compounds were determined with double digit nanomolar binding affinity, as well as similar potency in functional FMN competition assay. These results demonstrated the feasibility of small molecule identification against RNA targets using DEL selection. The developed experimental and computational strategy provided a promising opportunity for RNA ligand screening and expanded the application of DEL selection to a much wider context in drug discovery.

Reduced Flavin in Aqueous Solution Is Nonfluorescent.

Flavins are blue-light-absorbing chromophores with rich redox activity. Biologically, the most important are riboflavin (vitamin B2), flavin mononucleotide, and flavin adenine dinucleotide, the latter two of which are catalytic cofactors in enzymes. Flavins pivot between oxidized, one electron-, and two electron-reduced forms in different protonation states, depending on enzymatic requirements. Some flavoenzymes use light as a reagent for chemical bond formation, photoinduced electron transfer, or conformational changes required for light-sensitive signaling. Therefore, the photochemistry and photophysics of flavins have received wide attention. Fluorescence from oxidized flavin is often used to detect and track changes in flavin oxidation states. However, there have been conflicting reports over the past 45 years as to whether reduced flavin in solution has detectable fluorescence. Here, using single photon counting emission spectroscopy with rigorous sample preparation, we show definitively that reduced flavins are essentially nonfluorescent, having a quantum yield more than three orders of magnitude lower than oxidized flavin. This result will force a re-evaluation of experiments and models that assumed otherwise.

Structural and biochemical characterization of the prenylated flavin mononucleotide-dependent indole-3-carboxylic acid decarboxylase.

The ubiquitous UbiD family of reversible decarboxylases is implicated in a wide range of microbial processes and depends on the prenylated flavin mononucleotide cofactor for catalysis. However, only a handful of UbiD family members have been characterized in detail, and comparison between these has suggested considerable variability in enzyme dynamics and mechanism linked to substrate specificity. In this study, we provide structural and biochemical insights into the indole-3-carboxylic acid decarboxylase, representing an UbiD enzyme activity distinct from those previously studied. Structural insights from crystal structure determination combined with small-angle X-ray scattering measurements reveal that the enzyme likely undergoes an open-closed transition as a consequence of domain motion, an event that is likely coupled to catalysis. We also demonstrate that the indole-3-carboxylic acid decarboxylase can be coupled with carboxylic acid reductase to produce indole-3-carboxyaldehyde from indole + CO2 under ambient conditions. These insights provide further evidence for a common mode of action in the widespread UbiD enzyme family.

A novel formamidase is required for riboflavin biosynthesis in invasive bacteria.

Biosynthesis of riboflavin (RF), the precursor of the redox cofactors FMN and FAD, was thought to be well understood in bacteria, with all the pathway enzymes presumed to be known and essential. Our previous research has challenged this view by showing that, in the bacterium Sinorhizobium meliloti, deletion of the ribBA gene encoding the enzyme that catalyzes the initial steps on the RF biosynthesis pathway only causes a reduction in flavin secretion rather than RF auxotrophy. This finding led us to hypothesize that RibBA participates in the biosynthesis of flavins destined for secretion, whereas S. meliloti has another enzyme that performs this function for internal cellular metabolism. Here, we identify and biochemically characterize a novel formamidase (SMc02977) involved in the production of RF for intracellular functions in S. meliloti. This catalyst, which we named Sm-BrbF, releases formate from the early RF precursor 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate to yield 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate. We show that homologs of this enzyme are present in many bacteria, are highly abundant in the Rhizobiales order, and that sequence homologs from Brucella abortus and Liberobacter solanacearum complement the RF auxotrophy of the Sm1021ΔSMc02977 mutant. Furthermore, we show that the B. abortus enzyme (Bab2_0247, Ba-BrbF) is also an 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate formamidase, and that the bab2_0247 mutant is a RF auxotroph exhibiting a lower level of intracellular infection than the wildtype strain. Finally, we show that Sm-BrbF and Ba-BrbF directly interact with other RF biosynthesis pathway enzymes. Together, our results provide novel insight into the intricacies of RF biosynthesis in bacteria.

Sequential hydrolysis of FAD by ecto-5' nucleotidase CD73 and alkaline phosphatase is required for uptake of vitamin B2 into cells.

Extracellular hydrolysis of flavin-adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to riboflavin is thought to be important for cellular uptake of vitamin B2 because FAD and FMN are hydrophilic and do not pass the plasma membrane. However, it is not clear whether FAD and FMN are hydrolyzed by cell surface enzymes for vitamin B2 uptake. Here, we show that in human cells, FAD, a major form of vitamin B2 in plasma, is hydrolyzed by CD73 (also called ecto-5' nucleotidase) to FMN. Then, FMN is hydrolyzed by alkaline phosphatase to riboflavin, which is efficiently imported into cells. We determined that this two-step hydrolysis process is impaired on the surface of glycosylphosphatidylinositol (GPI)-deficient cells due to the lack of these GPI-anchored enzymes. During culture of GPI-deficient cells with FAD or FMN, we found that hydrolysis of these forms of vitamin B2 was impaired, and intracellular levels of vitamin B2 were significantly decreased compared with those in GPI-restored cells, leading to decreased formation of vitamin B2-dependent pyridoxal 5'-phosphate and mitochondrial dysfunction. Collectively, these results suggest that inefficient uptake of vitamin B2 might account for mitochondrial dysfunction seen in some cases of inherited GPI deficiency.

A higher plant FAD synthetase is fused to an inactivated FAD pyrophosphatase.

The riboflavin derivatives FMN and flavin adenine dinucleotide (FAD) are critical cofactors for wide-ranging biological processes across all kingdoms of life. Although it is well established that these flavins can be readily interconverted, in plants, the responsible catalysts and regulatory mechanisms remain poorly understood. Here, we report the cloning and biochemical characterization of an FAD synthetase encoded by the gene At5g03430, which we have designated AtFADS1 (A. thaliana FADS1). The catalytic properties of the FAD synthetase activity are similar to those reported for other FAD synthetases, except that we observed maximum activity with Zn2+ as the associated divalent metal cation. Like human FAD synthetase, AtFADS1 exists as an apparent fusion with an ancestral FAD pyrophosphatase, a feature that is conserved across plants. However, we detected no pyrophosphatase activity with AtFADS1, consistent with an observed loss of a key catalytic residue in higher plant evolutionary history. In contrast, we determined that algal FADS1 retains both FAD synthetase and pyrophosphatase activity. We discuss the implications, including the potential for yet-unstudied biologically relevant noncatalytic functions, and possible evolutionary pressures that have led to the loss of FAD pyrophosphatase activity, yet universal retention of an apparently nonfunctional domain in FADS of land plants.

In silico investigation on structure-function relationship of members belonging to the human SLC52 transporter family.

Riboflavin is an essential water-soluble vitamin that needs to be provided through the diet because of the conversion into flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), important cofactors in hundreds of flavoenzymes. The adsorption and distribution of riboflavin is mediated by transmembrane transporters of the SLC52 family, namely RFVT1-3, whose mutations are mainly associated with two diseases, MADD and the Brown-Vialetto-Van Laere syndrome. Interest in RFVTs as pharmacological targets has increased in the last few years due to their overexpression in several cancer cells, which can be exploited both by blocking the uptake of riboflavin into the cancerous cells, and by performing cancer targeted delivery of drugs with a high affinity for RFVTs. In this work, we propose three-dimensional structural models for all three human riboflavin transporters obtained by state-of-the-art artificial intelligence-based methods, which were then further refined with molecular dynamics simulations. Furthermore, two of the most notable mutations concerning RFVT2 and RFVT3 (W31S and N21S, respectively) were investigated studying the interactions between the wild-type and mutated transporters with riboflavin.