Obtaining the necessary molybdenum cofactor for sulfite oxidase activity in the nematode Caenorhabditis elegans surprisingly involves a dietary source.

Molybdenum cofactor (Moco) is a prosthetic group necessary for the activity of four unique enzymes, including the essential sulfite oxidase (SUOX-1). Moco is required for life; humans with inactivating mutations in the genes encoding Moco-biosynthetic enzymes display Moco deficiency, a rare and lethal inborn error of metabolism. Despite its importance to human health, little is known about how Moco moves among and between cells, tissues, and organisms. The prevailing view is that cells that require Moco must synthesize Moco de novo. Although, the nematode Caenorhabditis elegans appears to be an exception to this rule and has emerged as a valuable system for understanding fundamental Moco biology. C. elegans has the seemingly unique capacity to both synthesize its own Moco as well as acquire Moco from its microbial diet. However, the relative contribution of Moco from the diet or endogenous synthesis has not been rigorously evaluated or quantified biochemically. We genetically removed dietary or endogenous Moco sources in C. elegans and biochemically determined their impact on animal Moco content and SUOX-1 activity. We demonstrate that dietary Moco deficiency dramatically reduces both animal Moco content and SUOX-1 activity. Furthermore, these biochemical deficiencies have physiological consequences; we show that dietary Moco deficiency alone causes sensitivity to sulfite, the toxic substrate of SUOX-1. Altogether, this work establishes the biochemical consequences of depleting dietary Moco or endogenous Moco synthesis in C. elegans and quantifies the surprising contribution of the diet to maintaining Moco homeostasis in C. elegans.

Establishing pteridine metabolism in a progressive isogenic breast cancer cell model - part II.

INTRODUCTION: Determining the biological significance of pteridines in cancer development and progression remains an important step in understanding the altered levels of urinary pteridines seen in certain cancers. Our companion study revealed that several folate-derived pteridines and lumazines correlated with tumorigenicity in an isogenic, progressive breast cancer cell model, providing direct evidence for the tumorigenic origin of pteridines. OBJECTIVES: This study sought to elucidate the pteridine biosynthetic pathway in a progressive breast cancer model via direct pteridine dosing to determine how pteridine metabolism changes with tumorigenicity. METHODS: First, MCF10AT breast cancer cells were dosed individually with 15 pteridines to determine which pteridines were being metabolized and what metabolic products were being produced. Second, pteridines that were significantly metabolized were dosed individually across the progressive breast cancer cell model (MCF10A, MCF10AT, and MCF10ACA1a) to determine the relationship between each metabolic reaction and breast cancer tumorigenicity. RESULTS: Several pteridines were found to have altered metabolism in breast cancer cell lines, including pterin, isoxanthopterin, xanthopterin, sepiapterin, 6-biopterin, lumazine, and 7-hydroxylumazine (p < 0.05). In particular, isoxanthopterin and 6-biopterin concentrations were differentially expressed (p < 0.05) with respect to tumorigenicity following dosing with pterin and sepiapterin, respectively. Finally, the pteridine biosynthetic pathway in breast cancer cells was proposed based on these findings. CONCLUSIONS: This study, along with its companion study, demonstrates that pteridine metabolism becomes disrupted in breast cancer tumor cells. This work highlights several key metabolic reactions within the pteridine biosynthetic pathway that may be targeted for further investigation and clinical applications.

Discovery of Pteridine-7(8H)-one Derivatives as Potent and Selective Inhibitors of Bruton's Tyrosine Kinase (BTK).

Bruton's tyrosine kinase (BTK) is an attractive therapeutic target in the treatment of cancer, inflammation, and autoimmune diseases. Covalent and noncovalent BTK inhibitors have been developed, among which covalent BTK inhibitors have shown great clinical efficacy. However, some of them could produce adverse effects, such as diarrhea, rash, and platelet dysfunction, which are associated with the off-target inhibition of ITK and EGFR. In this study, we disclosed a series of pteridine-7(8H)-one derivatives as potent and selective covalent BTK inhibitors, which were optimized from 3z, an EGFR inhibitor previously reported by our group. Among them, compound 24a exhibited great BTK inhibition activity (IC50 = 4.0 nM) and high selectivity in both enzymatic (ITK >250-fold, EGFR >2500-fold) and cellular levels (ITK >227-fold, EGFR 27-fold). In U-937 xenograft models, 24a significantly inhibited tumor growth (TGI = 57.85%) at a 50 mg/kg dosage. Accordingly, 24a is a new BTK inhibitor worthy of further development.

A defect in molybdenum cofactor binding causes an attenuated form of sulfite oxidase deficiency.

Isolated sulfite oxidase deficiency (ISOD) is a rare recessive and infantile lethal metabolic disorder, which is caused by functional loss of sulfite oxidase (SO) due to mutations of the SUOX gene. SO is a mitochondrially localized molybdenum cofactor (Moco)- and heme-dependent enzyme, which catalyzes the vital oxidation of toxic sulfite to sulfate. Accumulation of sulfite and sulfite-related metabolites such as S-sulfocysteine (SSC) are drivers of severe neurodegeneration leading to early childhood death in the majority of ISOD patients. Full functionality of SO is dependent on correct insertion of the heme cofactor and Moco, which is controlled by a highly orchestrated maturation process. This maturation involves the translation in the cytosol, import into the intermembrane space (IMS) of mitochondria, cleavage of the mitochondrial targeting sequence, and insertion of both cofactors. Moco insertion has proven as the crucial step in this maturation process, which enables the correct folding of the homodimer and traps SO in the IMS. Here, we report on a novel ISOD patient presented at 17 months of age carrying the homozygous mutation NM_001032386.2 (SUOX):c.1097G > A, which results in the expression of SO variant R366H. Our studies show that histidine substitution of Arg366, which is involved in coordination of the Moco-phosphate, causes a severe reduction in Moco insertion efficacy in vitro and in vivo. Expression of R366H in HEK SUOX-/- cells mimics the phenotype of patient's fibroblasts, representing a loss of SO expression and specific activity. Our studies disclose a general paradigm for a kinetic defect in Moco insertion into SO caused by residues involved in Moco coordination resulting in the case of R366H in an attenuated form of ISOD.

A-Type Carrier Proteins Are Involved in [4Fe-4S] Cluster Insertion into the Radical S-Adenosylmethionine Protein MoaA for the Synthesis of Active Molybdoenzymes.

Iron sulfur (Fe-S) clusters are important biological cofactors present in proteins with crucial biological functions, from photosynthesis to DNA repair, gene expression, and bioenergetic processes. For the insertion of Fe-S clusters into proteins, A-type carrier proteins have been identified. So far, three of them have been characterized in detail in Escherichia coli, namely, IscA, SufA, and ErpA, which were shown to partially replace each other in their roles in [4Fe-4S] cluster insertion into specific target proteins. To further expand the knowledge of [4Fe-4S] cluster insertion into proteins, we analyzed the complex Fe-S cluster-dependent network for the synthesis of the molybdenum cofactor (Moco) and the expression of genes encoding nitrate reductase in E. coli. Our studies include the identification of the A-type carrier proteins ErpA and IscA, involved in [4Fe-4S] cluster insertion into the radical S-adenosyl-methionine (SAM) enzyme MoaA. We show that ErpA and IscA can partially replace each other in their role to provide [4Fe-4S] clusters for MoaA. Since most genes expressing molybdoenzymes are regulated by the transcriptional regulator for fumarate and nitrate reduction (FNR) under anaerobic conditions, we also identified the proteins that are crucial to obtain an active FNR under conditions of nitrate respiration. We show that ErpA is essential for the FNR-dependent expression of the narGHJI operon, a role that cannot be compensated by IscA under the growth conditions tested. SufA does not appear to have a role in Fe-S cluster insertion into MoaA or FNR under anaerobic growth employing nitrate respiration, based on the low level of gene expression. IMPORTANCE Understanding the assembly of iron-sulfur (Fe-S) proteins is relevant to many fields, including nitrogen fixation, photosynthesis, bioenergetics, and gene regulation. Remaining critical gaps in our knowledge include how Fe-S clusters are transferred to their target proteins and how the specificity in this process is achieved, since different forms of Fe-S clusters need to be delivered to structurally highly diverse target proteins. Numerous Fe-S carrier proteins have been identified in prokaryotes like Escherichia coli, including ErpA, IscA, SufA, and NfuA. In addition, the diverse Fe-S cluster delivery proteins and their target proteins underlie a complex regulatory network of expression, to ensure that both proteins are synthesized under particular growth conditions.

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.

Site-Directed Mutagenesis at the Molybdenum Pterin Cofactor Site of the Human Aldehyde Oxidase: Interrogating the Kinetic Differences Between Human and Cynomolgus Monkey.

The estimation of the drug clearance by aldehyde oxidase (AO) has been complicated because of this enzyme's atypical kinetics and species and substrate specificity. Since human AO (hAO) and cynomolgus monkey AO (mAO) have a 95.1% sequence identity, cynomolgus monkeys may be the best species for estimating AO clearance in humans. Here, O6-benzylguanine (O6BG) and dantrolene were used under anaerobic conditions, as oxidative and reductive substrates of AO, respectively, to compare and contrast the kinetics of these two species through numerical modeling. Whereas dantrolene reduction followed the same linear kinetics in both species, the oxidation rate of O6BG was also linear in mAO and did not follow the already established biphasic kinetics of hAO. In an attempt to determine why hAO and mAO are kinetically distinct, we have altered the hAO V811 and F885 amino acids at the oxidation site adjacent to the molybdenum pterin cofactor to the corresponding alanine and leucine in mAO, respectively. Although some shift to a more monkey-like kinetics was observed for the V811A mutant, five more mutations around the AO cofactors still need to be investigated for this purpose. In comparing the oxidative and reductive rates of metabolism under anaerobic conditions, we have come to the conclusion that despite having similar rates of reduction (4-fold difference), the oxidation rate in mAO is more than 50-fold slower than hAO. This finding implies that the presence of nonlinearity in AO kinetics is dependent upon the degree of imbalance between the rates of oxidation and reduction in this enzyme. SIGNIFICANCE STATEMENT: Although they have as much as 95.1% sequence identity, human and cynomolgus monkey aldehyde oxidase are kinetically distinct. Therefore, monkeys may not be good estimators of drug clearance in humans.

Identification and characterisation of the Volvox carteri Moco carrier protein.

The molybdenum cofactor (Moco) is a redox active prosthetic group found in the active site of Moco-dependent enzymes (Mo-enzymes). As Moco and its intermediates are highly sensitive towards oxidative damage, these are believed to be permanently protein bound during synthesis and upon maturation. As a major component of the plant Moco transfer and storage system, proteins have been identified that are capable of Moco binding and release but do not possess Moco-dependent enzymatic activities. The first protein found to possess these properties was the Moco carrier protein (MCP) from the green alga Chlamydomonas reinhardtii. Here, we describe the identification and biochemical characterisation of the Volvox carteri (V. carteri) MCP and, for the first time, employ a comparative analysis to elucidate the principles behind MCP Moco binding. Doing so identified a sequence region of low homology amongst the existing MCPs, which we showed to be essential for Moco binding to V. carteri MCP.

Age determination of the adult blow fly Lucilia sericata (Diptera: Calliphoridae) through quantitative pteridine fluorescence analysis.

Determination of a minimal postmortem interval via age estimation of necrophagous diptera has been restricted to the juvenile stages and the time until emergence of the adult fly, i.e. up until 2-6 weeks depending on species and temperature. Age estimation of adult flies could extend this period by adding the age of the fly to the time needed for complete development. In this context pteridines are promising metabolites, as they accumulate in the eyes of flies with increasing age. We studied adults of the blow fly Lucilia sericata at constant temperatures of 16 °C and 25 °C up to an age of 25 days and estimated their pteridine levels by fluorescence spectroscopy. Age was given in accumulated degree days (ADD) across temperatures. Additionally, a mock case was set up to test the applicability of the method. Pteridine increases logarithmically with increasing ADD, but after 70-80 ADD the increase slows down and the curve approaches a maximum. Sex had a significant impact (p < 4.09 × 10-6) on pteridine fluorescence level, while body-size and head-width did not. The mock case demonstrated that a slight overestimation of the real age (in ADD) only occurred in two out of 30 samples. Age determination of L. sericata on the basis of pteridine levels seems to be limited to an age of about 70 ADD, but depending on the ambient temperature this could cover an extra amount of time of about 5-7 days after completion of the metamorphosis.

The structure of the Moco carrier protein from Rippkaea orientalis.

The molybdenum cofactor (Moco) is the prosthetic group of all molybdenum-dependent enzymes except for nitrogenase. The multistep biosynthesis pathway of Moco and its function in molybdenum-dependent enzymes are already well understood. The mechanisms of Moco transfer, storage and insertion, on the other hand, are not. In the cell, Moco is usually not found in its free form and remains bound to proteins because of its sensitivity to oxidation. The green alga Chlamydomonas reinhardtii harbors a Moco carrier protein (MCP) that binds and protects Moco but is devoid of enzymatic function. It has been speculated that this MCP acts as a means of Moco storage and transport. Here, the search for potential MCPs has been extended to the prokaryotes, and many MCPs were found in cyanobacteria. A putative MCP from Rippkaea orientalis (RoMCP) was selected for recombinant production, crystallization and structure determination. RoMCP has a Rossmann-fold topology that is characteristic of nucleotide-binding proteins and a homotetrameric quaternary structure similar to that of the MCP from C. reinhardtii. In each protomer, a positively charged crevice was identified that accommodates up to three chloride ions, hinting at a potential Moco-binding site. Computational docking experiments supported this notion and gave an impression of the RoMCP-Moco complex.

Sulfamethoxazole drug stress upregulates antioxidant immunomodulatory metabolites in Escherichia coli.

Escherichia coli is an important model organism in microbiology and a prominent member of the human microbiota1. Environmental isolates readily colonize the gastrointestinal tract of humans and other animals, and they can serve diverse probiotic, commensal and pathogenic roles in the host2-4. Although certain strains have been associated with the severity of inflammatory bowel disease (IBD)2,5, the diverse immunomodulatory phenotypes remain largely unknown at the molecular level. Here, we decode a previously unknown E. coli metabolic pathway that produces a family of hybrid pterin-phenylpyruvate conjugates, which we named the colipterins. The metabolites are upregulated by subinhibitory levels of the antifolate sulfamethoxazole, which is used to treat infections including in patients with IBD6,7. The genes folX/M and aspC/tyrB involved in monapterin biosynthesis8-10 and aromatic amino acid transamination,11 respectively, were required to initiate the colipterin pathway. We show that the colipterins are antioxidants, harbour diverse immunological activities in primary human tissues, activate anti-inflammatory interleukin-10 and improve colitis symptoms in a colitis mouse model. Our study defines an antifolate stress response in E. coli and links its associated metabolites to a major immunological marker of IBD.

Identification and age-dependence of pteridines in bed bugs (Cimex lectularius) and bat bugs (C. pipistrelli) using liquid chromatography-tandem mass spectrometry.

Determining the age of free-living insects, particularly of blood-sucking species, is important for human health because such knowledge critically influences the estimates of biting frequency and vectoring ability. Genetic age determination is currently not available. Pteridines gradually accumulate in the eyes of insects and their concentrations is the prevailing method. Despite of their stability, published extractions differ considerably, including for standards, for mixtures of pteridines and even for light conditions. This methodological inconsistency among studies is likely to influence age estimates severely and to hamper their comparability. Therefore we reviewed methodological steps across 106 studies to identify methodological denominators and results across studies. Second, we experimentally test how different pteridines vary in their age calibration curves in, common bed (Cimex lectularius) and bat bugs (C. pipistrelli). Here we show that the accumulation of particular pteridines varied between a) different populations and b) rearing temperatures but not c) with the impact of light conditions during extraction or d) the type of blood consumed by the bugs. To optimize the extraction of pteridines and measuring concentrations, we recommend the simultaneous measurement of more than one standard and subsequently to select those that show consistent changes over time to differentiate among age cohorts.

Being red, blue and green: the genetic basis of coloration differences in the strawberry poison frog (Oophaga pumilio).

BACKGROUND: Animal coloration is usually an adaptive attribute, under strong local selection pressures and often diversified among species or populations. The strawberry poison frog (Oophaga pumilio) shows an impressive array of color morphs across its distribution in Central America. Here we quantify gene expression and genetic variation to identify candidate genes involved in generating divergence in coloration between populations of red, green and blue O. pumilio from the Bocas del Toro archipelago in Panama. RESULTS: We generated a high quality non-redundant reference transcriptome by mapping the products of genome-guided and de novo transcriptome assemblies onto a re-scaffolded draft genome of O. pumilio. We then measured gene expression in individuals of the three color phenotypes and identified color-associated candidate genes by comparing differential expression results against a list of a priori gene sets for five different functional categories of coloration - pteridine synthesis, carotenoid synthesis, melanin synthesis, iridophore pathways (structural coloration), and chromatophore development. We found 68 candidate coloration loci with significant expression differences among the color phenotypes. Notable upregulated examples include pteridine synthesis genes spr, xdh and pts (in red and green frogs); carotenoid metabolism genes bco2 (in blue frogs), scarb1 (in red frogs), and guanine metabolism gene psat1 (in blue frogs). We detected significantly higher expression of the pteridine synthesis gene set in red and green frogs versus blue frogs. In addition to gene expression differences, we identified 370 outlier SNPs on 162 annotated genes showing signatures of diversifying selection, including eight pigmentation-associated genes. CONCLUSIONS: Gene expression in the skin of the three populations of frogs with differing coloration is highly divergent. The strong signal of differential expression in pteridine genes is consistent with a major role of these genes in generating the coloration differences among the three morphs. However, the finding of differentially expressed genes across pathways and functional categories suggests that multiple mechanisms are responsible for the coloration differences, likely involving both pigmentary and structural coloration. In addition to regulatory differences, we found potential evidence of differential selection acting at the protein sequence level in several color-associated loci, which could contribute to the color polymorphism.

A recently evolved diflavin-containing monomeric nitrate reductase is responsible for highly efficient bacterial nitrate assimilation.

Nitrate is one of the major inorganic nitrogen sources for microbes. Many bacterial and archaeal lineages have the capacity to express assimilatory nitrate reductase (NAS), which catalyzes the rate-limiting reduction of nitrate to nitrite. Although a nitrate assimilatory pathway in mycobacteria has been proposed and validated physiologically and genetically, the putative NAS enzyme has yet to be identified. Here, we report the characterization of a novel NAS encoded by Mycolicibacterium smegmatis Msmeg_4206, designated NasN, which differs from the canonical NASs in its structure, electron transfer mechanism, enzymatic properties, and phylogenetic distribution. Using sequence analysis and biochemical characterization, we found that NasN is an NADPH-dependent, diflavin-containing monomeric enzyme composed of a canonical molybdopterin cofactor-binding catalytic domain and an FMN-FAD/NAD-binding, electron-receiving/transferring domain, making it unique among all previously reported hetero-oligomeric NASs. Genetic studies revealed that NasN is essential for aerobic M. smegmatis growth on nitrate as the sole nitrogen source and that the global transcriptional regulator GlnR regulates nasN expression. Moreover, unlike the NADH-dependent heterodimeric NAS enzyme, NasN efficiently supports bacterial growth under nitrate-limiting conditions, likely due to its significantly greater catalytic activity and oxygen tolerance. Results from a phylogenetic analysis suggested that the nasN gene is more recently evolved than those encoding other NASs and that its distribution is limited mainly to Actinobacteria and Proteobacteria. We observed that among mycobacterial species, most fast-growing environmental mycobacteria carry nasN, but that it is largely lacking in slow-growing pathogenic mycobacteria because of multiple independent genomic deletion events along their evolution.

Anion Binding and Oxidative Modification at the Molybdenum Cofactor of Formate Dehydrogenase from Rhodobacter capsulatus Studied by X-ray Absorption Spectroscopy.

Formate dehydrogenase (FDH) enzymes are versatile catalysts for CO2 conversion. The FDH from Rhodobacter capsulatus contains a molybdenum cofactor with the dithiolene functions of two pyranopterin guanine dinucleotide molecules, a conserved cysteine, and a sulfido group bound at Mo(VI). In this study, we focused on metal oxidation state and coordination changes in response to exposure to O2, inhibitory anions, and redox agents using X-ray absorption spectroscopy (XAS) at the Mo K-edge. Differences in the oxidative modification of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor relative to samples prepared aerobically without inhibitor, such as variations in the relative numbers of sulfido (Mo═S) and oxo (Mo═O) bonds, were observed in the presence of azide (N3-) or cyanate (OCN-). Azide provided best protection against O2, resulting in a quantitatively sulfurated cofactor with a displaced cysteine ligand and optimized formate oxidation activity. Replacement of the cysteine ligand by a formate (HCO2-) ligand at the molybdenum in active enzyme is compatible with our XAS data. Cyanide (CN-) inactivated the enzyme by replacing the sulfido ligand at Mo(VI) with an oxo ligand. Evidence that the sulfido group may become protonated upon molybdenum reduction was obtained. Our results emphasize the role of coordination flexibility at the molybdenum center during inhibitory and catalytic processes of FDH enzymes.

Insights into the Cnx1E catalyzed MPT-AMP hydrolysis.

Molybdenum insertases (Mo-insertases) catalyze the final step of molybdenum cofactor (Moco) biosynthesis, an evolutionary old and highly conserved multi-step pathway. In the first step of the pathway, GTP serves as substrate for the formation of cyclic pyranopterin monophosphate, which is subsequently converted into molybdopterin (MPT) in the second pathway step. In the following synthesis steps, MPT is adenylated yielding MPT-AMP that is subsequently used as substrate for enzyme catalyzed molybdate insertion. Molybdate insertion and MPT-AMP hydrolysis are catalyzed by the Mo-insertase E-domain. Earlier work reported a highly conserved aspartate residue to be essential for Mo-insertase functionality. In this work, we confirmed the mechanistic relevance of this residue for the Arabidopsis thaliana Mo-insertase Cnx1E. We found that the conservative substitution of Cnx1E residue Asp274 by Glu (D274E) leads to an arrest of MPT-AMP hydrolysis and hence to the accumulation of MPT-AMP. We further showed that the MPT-AMP accumulation goes in hand with the accumulation of molybdate. By crystallization and structure determination of the Cnx1E variant D274E, we identified the potential reason for the missing hydrolysis activity in the disorder of the region spanning amino acids 269 to 274. We reasoned that this is caused by the inability of a glutamate in position 274 to coordinate the octahedral Mg2+-water complex in the Cnx1E active site.

Identification and Characterization of a New Class of (6-4) Photolyase from Vibrio cholerae.

Light is crucial for many biological activities of most organisms, including vision, resetting of circadian rhythm, photosynthesis, and DNA repair. The cryptochrome/photolyase family (CPF) represents an ancient group of UV-A/blue light sensitive proteins that perform different functions such as DNA repair, circadian photoreception, and transcriptional regulation. The CPF is widely distributed throughout all organisms, including marine prokaryotes. The bacterium Vibrio cholerae was previously shown to have a CPD photolyase that repairs UV-induced thymine dimers and two CRY-DASHs that repair UV-induced single-stranded DNA damage. Here, we characterize a hypothetical gene Vca0809 encoding a new member of CPF in this organism. The spectroscopic analysis of the purified protein indicated that this enzyme possessed a catalytic cofactor, FAD, and photoantenna chromophore 6,7-dimethyl 8-ribityl-lumazin. With a slot blot-based DNA repair assay, we showed that it possessed (6-4) photolyase activity. Further phylogenetic and computational analyses enabled us to classify this gene as a member of the family of iron-sulfur bacterial cryptochromes and photolyases (FeS-BCP). Therefore, we named this gene Vc(6-4) FeS-BCP.

The regulation of Moco biosynthesis and molybdoenzyme gene expression by molybdenum and iron in bacteria.

Bacterial molybdoenzymes are key enzymes involved in the global sulphur, nitrogen and carbon cycles. These enzymes require the insertion of the molybdenum cofactor (Moco) into their active sites and are able to catalyse a large range of redox-reactions. Escherichia coli harbours nineteen different molybdoenzymes that require a tight regulation of their synthesis according to substrate availability, oxygen availability and the cellular concentration of molybdenum and iron. The synthesis and assembly of active molybdoenzymes are regulated at the level of transcription of the structural genes and of translation in addition to the genes involved in Moco biosynthesis. The action of global transcriptional regulators like FNR, NarXL/QP, Fur and ArcA and their roles on the expression of these genes is described in detail. In this review we focus on what is known about the molybdenum- and iron-dependent regulation of molybdoenzyme and Moco biosynthesis genes in the model organism E. coli. The gene regulation in E. coli is compared to two other well studied model organisms Rhodobacter capsulatus and Shewanella oneidensis.

Cooption of the pteridine biosynthesis pathway underlies the diversification of embryonic colors in water striders.

Naturalists have been fascinated for centuries by animal colors and color patterns. While widely studied at the adult stage, we know little about color patterns in the embryo. Here, we study a trait consisting of coloration that is specific to the embryo and absent from postembryonic stages in water striders (Gerromorpha). By combining developmental genetics with chemical and phylogenetic analyses across a broad sample of species, we uncovered the mechanisms underlying the emergence and diversification of embryonic colors in this group of insects. We show that the pteridine biosynthesis pathway, which ancestrally produces red pigment in the eyes, has been recruited during embryogenesis in various extraocular tissues including antennae and legs. In addition, we discovered that this cooption is common to all water striders and initially resulted in the production of yellow extraocular color. Subsequently, 6 lineages evolved bright red color and 2 lineages lost the color independently. Despite the high diversity in colors and color patterns, we show that the underlying biosynthesis pathway remained stable throughout the 200 million years of Gerromorpha evolutionary time. Finally, we identified erythropterin and xanthopterin as the pigments responsible for these colors in the embryo of various species. These findings demonstrate how traits can emerge through the activation of a biosynthesis pathway in new developmental contexts.

Deconvolution of reduction potentials of formate dehydrogenase from Cupriavidus necator.

The formate dehydrogenase enzyme from Cupriavidus necator (FdsABG) carries out the two-electron oxidation of formate to CO2, but is also capable of reducing CO2 back to formate, a potential biofuel. FdsABG is a heterotrimeric enzyme that performs this transformation using nine redox-active cofactors: a bis(molybdopterin guanine dinucleotide) (bis-MGD) at the active site coupled to seven iron-sulfur clusters, and one equivalent of flavin mononucleotide (FMN). To better understand the pathway of electron flow in FdsABG, the reduction potentials of the various cofactors were examined through direct electrochemistry. Given the redundancy of cofactors, a truncated form of the FdsA subunit was developed that possesses only the bis-MGD active site and a singular [4Fe-4S] cluster. Electrochemical characterization of FdsABG compared to truncated FdsA shows that the measured reduction potentials are remarkably similar despite the truncation with two observable features at - 265 mV and - 455 mV vs SHE, indicating that the voltammetry of the truncated enzyme is representative of the reduction potentials of the intact heterotrimer. By producing truncated FdsA without the necessary maturation factors required for bis-MGD insertion, a form of the truncated FdsA that possesses only the [4Fe-4S] was produced, which gives a single voltammetric feature at - 525 mV, allowing the contributions of the molybdenum cofactor to be associated with the observed feature at - 265 mV. This method allowed for the deconvolution of reduction potentials for an enzyme with highly complex cofactor content to know more about the thermodynamic landscape of catalysis.