A conserved and seemingly redundant Escherichia coli biotin biosynthesis gene expressed only during anaerobic growth.

Biotin is an essential metabolic cofactor and de novo biotin biosynthetic pathways are widespread in microorganisms and plants. Biotin synthetic genes are generally found clustered into bio operons to facilitate tight regulation since biotin synthesis is a metabolically expensive process. Dethiobiotin synthetase (DTBS) catalyzes the penultimate step of biotin biosynthesis, the formation of 7,8-diaminononanoate (DAPA). In Escherichia coli, DTBS is encoded by the bio operon gene bioD. Several studies have reported transcriptional activation of ynfK a gene of unknown function, under anaerobic conditions. Alignments of YnfK with BioD have led to suggestions that YnfK has DTBS activity. We report that YnfK is a functional DTBS, although an enzyme of poor activity that is poorly expressed. Supplementation of growth medium with DAPA or substitution of BioD active site residues for the corresponding YnfK residues greatly improved the DTBS activity of YnfK. We confirmed that FNR activates transcriptional level of ynfK during anaerobic growth and identified the FNR binding site of ynfK. The ynfK gene is well conserved in γ-proteobacteria.

Efficient induction of proximity-dependent labelling by biotin feeding in BMAL1-BioID knock-in mice.

Proximity-dependent biotin identification (BioID) is a useful method to identify unknown protein-protein interactions. Few reports have described genetically engineered knock-in mouse models for in vivo BioID. Thus, little is known about the proper method for biotin administration and which tissues are applicable. Here, we established a BioID knock-in mouse model of Brain and Muscle ARNT-Like 1 (BMAL1) and the BirA biotin ligase with R118G mutation (BirA*). The BMAL1-BioID mouse model was used to investigate the effect of biotin diet feeding on protein biotinylation in several tissues. The BMAL1-BirA* fusion protein-retained proper intracellular localization of BMAL1 and binding to CLOCK protein in HEK293T cells. A biotin labelling assay in mouse embryonic fibroblasts revealed the protein biotinylation activity of BMAL1-BirA* expressed in knock-in mouse cells depending on biotin supplementation. Lastly, feeding a 0.5% biotin diet for 7 days induced protein biotinylation in the brain, heart, testis and liver of BMAL1-BioID mice without adverse effects on spermatogenesis. In the kidney, the biotin diet increased biotinylated protein levels in BMAL1-BioID and control mice, suggesting the existence of endogenous biotinylation activity. These results provide valuable information to optimize the in vivo BioID procedure.

A new bivalent fluorescent fusion protein for differential Cu(II) and Zn(II) ion detection in aqueous solution.

Simple and easy to engineer metal-sensing molecules that are capable of differentiating metal ions and producing metal-specific signals are highly desirable. Metal ions affect the thermal stability of proteins by increasing or decreasing their resistance to unfolding. This work illustrates a new strategy for designing bivalent fluorescent fusion proteins capable of differentiating metal ions in solution through their distinct effects on a protein's thermal stability. A new dual purpose metal sensor was developed consisting of biotin protein ligase (BirA) from B. pseudomallei (Bp) fused to green fluorescent protein (GFP). When coupled with differential scanning fluorimetry of GFP-tagged proteins (DSF-GTP) for signal-transduction detection, Bp BirA-GFP yields distinct protein unfolding signatures with Zn(II) and Cu(II) ions in aqueous solutions. The limit of detection of the system is ∼1 µM for both metal species. The system can be used in a variety of high-throughput assay formats including for the screening of metal-binding proteins and chelators. Bp BirA-GFP has also the additional benefit of being useful in Cu(II) ion field-testing applications through simple visual observation of a temperature-dependent loss of fluorescence. Bp BirA-GFP is the first example of a 2protein-based dual purpose Cu(II) and Zn(II) ion sensor compatible with two different yet complementary signal-transduction detection systems.

Efficient fermentative production of L-theanine by Corynebacterium glutamicum.

L-Theanine is a unique non-protein amino acid found in tea plants that has been shown to possess numerous functional properties relevant to food science and human nutrition. L-Theanine has been commercially developed as a valuable additive for use in food and beverages, and its market is expected to expand substantially if the production cost can be lowered. Although the enzymatic approach holds considerable potential for use in L-theanine production, demand exists for developing more tractable methods (than those currently available) that can be implemented under mild conditions and will reduce operational procedures and cost. Here, we sought to engineer fermentative production of L-theanine in Corynebacterium glutamicum, an industrially safe host. For L-theanine synthesis, we used γ-glutamylmethylamide synthetase (GMAS), which catalyzes the ATP-dependent ligation of L-glutamate and ethylamine. First, distinct GMASs were expressed in C. glutamicum wild-type ATCC 13032 strain and GDK-9, an L-glutamate overproducing strain, to produce L-theanine upon ethylamine addition to the hosts. Second, the L-glutamate exporter in host cells was disrupted, which markedly increased the L-theanine titer in GDK-9 cells and almost eliminated the accumulation of L-glutamate in the culture medium. Third, a chromosomally gmasMm-integrated L-alanine producer was constructed and used, attempting to synthesize ethylamine endogenously by expressing plant-derived L-serine/L-alanine decarboxylases; however, these enzymes showed no L-alanine decarboxylase activity under our experimental conditions. The optimal engineered strain that we ultimately created produced ~ 42 g/L L-theanine, with a yield of 19.6%, in a 5-L fermentor. This is the first report of fermentative production of L-theanine achieved using ethylamine supplementation.

Citryl Ornithine Is an Intermediate in a Three-Step Biosynthetic Pathway for Rhizoferrin in Francisella.

The Gram-negative bacterium Francisella tularensis secretes the siderophore rhizoferrin to scavenge necessary iron from the environment. Rhizoferrin, also produced by a variety of fungi and bacteria, comprises two citrate molecules linked by amide bonds to a central putrescine (diaminobutane) moiety. Genetic analysis has determined that rhizoferrin production in F. tularensis requires two enzymes: FslA, a siderophore synthetase of the nonribosomal peptide synthetase-independent siderophore synthetase (NIS) family, and FslC, a pyridoxal-phosphate-dependent decarboxylase. To discern the steps in the biosynthetic pathway, we tested F. tularensis strain LVS and its ΔfslA and ΔfslC mutants for the ability to incorporate potential precursors into rhizoferrin. Unlike putrescine supplementation, supplementation with ornithine greatly enhanced siderophore production by LVS. Radioactivity from L-[U-14C] ornithine, but not from L-[1-14C] ornithine, was efficiently incorporated into rhizoferrin by LVS. Although neither the ΔfslA nor the ΔfslC mutant produced rhizoferrin, a putative siderophore intermediate labeled by both [U-14C] ornithine and [1-14C] ornithine was secreted by the ΔfslC mutant. Rhizoferrin was identified by liquid chromatography and mass spectrometry in LVS culture supernatants, while citryl-ornithine was detected as the siderophore intermediate in the culture supernatant of the ΔfslC mutant. Our findings support a three-step pathway for rhizoferrin production in Francisella; unlike the fungus Rhizopus delemar, where putrescine functions as a primary precursor for rhizoferrin, biosynthesis in Francisella preferentially starts with ornithine as the substrate for FslA-mediated condensation with citrate. Decarboxylation of this citryl ornithine intermediate by FslC is necessary for a second condensation reaction with citrate to produce rhizoferrin.

Proximity-dependent biotinylation mediated by TurboID to identify protein-protein interaction networks in yeast.

The use of proximity-dependent biotinylation assays coupled to mass spectrometry (PDB-MS) has changed the field of protein-protein interaction studies. However, despite the recurrent and successful use of BioID-based protein-protein interactions screening in mammalian cells, the implementation of PDB-MS in yeast has not been effective. Here, we report a simple and rapid approach in yeast to effectively screen for proximal and interacting proteins in their natural cellular environment by using TurboID, a recently described version of the BirA biotin ligase. Using the protein arginine methyltransferase Rmt3 and the RNA exosome subunits, Rrp6 and Dis3, the application of PDB-MS in yeast by using TurboID was able to recover protein-protein interactions previously identified using other biochemical approaches and provided new complementary information for a given protein bait. The development of a rapid and effective PDB assay that can systematically analyze protein-protein interactions in living yeast cells opens the way for large-scale proteomics studies in this powerful model organism.

Antisense RNA-Based Strategy for Enhancing Surfactin Production in Bacillus subtilis TS1726 via Overexpression of the Unconventional Biotin Carboxylase II To Enhance ACCase Activity.

The antisense RNA (asRNA) strategy is commonly used to block protein expression and downregulate the contents of metabolites in several microorganisms. Here, we show that the asRNA strategy can also be used to block gfp expression in Bacillus subtilis TS1726, which could further be utilized in the identification of new genes and functions. Via application of this strategy, biotin carboxylase II encoded by yngH (GeneID 939474) was identified to play a more significant role in maintaining acetyl-CoA carboxylase (ACCase) activity and enhancing surfactin synthesis compared to those of other ACCase subunits. The yngH gene was then overexpressed in the engineered strain B. subtilis TS1726(yngH). The surfactin titer of TS1726(yngH) increased to 13.37 g/L in a flask culture, representing a 43% increase compared to that of parental strain TS1726. This strategy opens the door to achieving large-scale production and broad application of surfactin.

A platform for discovery of functional cell-penetrating peptides for efficient multi-cargo intracellular delivery.

Cell penetrating peptides (CPPs) offer great potential to deliver therapeutic molecules to previously inaccessible intracellular targets. However, many CPPs are inefficient and often leave their attached cargo stranded in the cell's endosome. We report a versatile platform for the isolation of peptides delivering a wide range of cargos into the cytoplasm of cells. We used this screening platform to identify multiple "Phylomer" CPPs, derived from bacterial and viral genomes. These peptides are amenable to conventional sequence optimization and engineering approaches for cell targeting and half-life extension. We demonstrate potent, functional delivery of protein, peptide, and nucleic acid analog cargos into cells using Phylomer CPPs. We validate in vivo activity in the cytoplasm, through successful transport of an oligonucleotide therapeutic fused to a Phylomer CPP in a disease model for Duchenne's muscular dystrophy. This report thus establishes a discovery platform for identifying novel, functional CPPs to expand the delivery landscape of druggable intracellular targets for biological therapeutics.

Biotin-mediated growth and gene expression in Staphylococcus aureus is highly responsive to environmental biotin.

Biotin (Vitamin B7) is a critical enzyme co-factor in metabolic pathways important for bacterial survival. Biotin is obtained either from the environment or by de novo synthesis, with some bacteria capable of both. In certain species, the bifunctional protein BirA plays a key role in biotin homeostasis as it regulates expression of biotin biosynthetic enzymes in response to biotin demand and supply. Here, we compare the effect of biotin on the growth of two bacteria that possess a bifunctional BirA, namely Escherichia coli and Staphylococcus aureus. Unlike E. coli that could fulfill its biotin requirements through de novo synthesis, S. aureus showed improved growth rates in media supplemented with 10 nM biotin. S. aureus also accumulated more radiolabeled biotin from the media highlighting its ability to efficiently scavenge exogenous material. These data are consistent with S. aureus colonizing low biotin microhabitats. We also demonstrate that the S. aureus BirA protein is a transcriptional repressor of BioY, a subunit of the biotin transporter, and an operon containing yhfT and yhfS, the products of which have a putative role in fatty acid homeostasis. Increased expression of bioY is proposed to help cue S. aureus for efficient scavenging in low biotin environments.

Analysis of SUMOylated Proteins in Cells and In Vivo Using the bioSUMO Strategy.

Posttranslational regulation of proteins by conjugation of ubiquitin- and ubiquitin-like molecules is a common theme in almost every known biological pathway. SUMO (small ubiquitin-related modifier) is dynamically added and deleted from many cellular substrates to control activity, localization, and recruitment of other SUMO-recognizing protein complexes. The dynamic nature of this modification and its low abundance in resting cells make it challenging to study, with susceptibility to deSUMOylases further complicating its analysis. Here we describe bioSUMO, a general method to isolate and analyze SUMOylated proteins from cultured cells, using Drosophila as a highlighted example. The method also has been validated in transgenic flies, as well as human cells. SUMOylated substrates are labeled by in vivo biotinylation, which facilitates their subsequent purification using streptavidin-based affinity chromatography under stringent conditions and with very low background. The bioSUMO approach can be used to validate whether a specific protein is modified, or used to analyze an entire SUMO subproteome. If coupled to quantitative proteomics methods, it may reveal how the SUMO landscape changes with different stimuli, or in diverse cell or tissue types. This technique offers a complementary approach to study SUMO biology and we expect that the strategy can be extended to other ubiquitin-like proteins.

An improved smaller biotin ligase for BioID proximity labeling.

The BioID method uses a promiscuous biotin ligase to detect protein-protein associations as well as proximate proteins in living cells. Here we report improvements to the BioID method centered on BioID2, a substantially smaller promiscuous biotin ligase. BioID2 enables more-selective targeting of fusion proteins, requires less biotin supplementation, and exhibits enhanced labeling of proximate proteins. Thus BioID2 improves the efficiency of screening for protein-protein associations. We also demonstrate that the biotinylation range of BioID2 can be considerably modulated using flexible linkers, thus enabling application-specific adjustment of the biotin-labeling radius.

Cell Lysis in S. pombe ura4 Mutants Is Suppressed by Loss of Functional Pub1, Which Regulates the Uracil Transporter Fur4.

Schizosaccharomyces pombe Δura4 cells lyse when grown on YPD medium. A S. pombe non-essential gene deletion library was screened to determine suppressors of the lysis phenotype. Deletion of the pub1 gene, which encoded E3 ubiquitin ligase, strongly suppressed cell lysis in Δura4 cells. The Δpub1 cells displayed high sensitivity to 5-fluorouracil, a toxic analog of uracil, and this sensitivity was suppressed by deletion of fur4, which encoded a uracil transporter. Fur4 localized primarily to the Golgi apparatus and vacuoles in wild-type cells, but localization was predominantly at the plasma membrane in Δpub1 cells. Fur4 was necessary for the utilization of extracellular uracil, cytosine, or UMP. Uracil uptake activity increased in the Δpub1 strain in a Fur4-dependent manner. In addition, uracil starvation was critical for induction of cell lysis of Δura4 strains and uracil supplementation suppressed lysis. In summary, the increased uracil uptake ability of Δpub1 cells, where Fur4 was predominantly localized to the plasma membrane, resulted in suppression of cell lysis in the Δura4 background.

Biotin deprivation impairs mitochondrial structure and function and has implications for inherited metabolic disorders.

Certain inborn errors of metabolism result from deficiencies in biotin containing enzymes. These disorders are mimicked by dietary absence or insufficiency of biotin, ATP deficit being a major effect,whose responsible mechanisms have not been thoroughly studied. Here we show that in rats and cultured cells it is the result of reduced TCA cycle flow, partly due to deficient anaplerotic biotin-dependent pyruvate carboxylase. This is accompanied by diminished flow through the electron transport chain, augmented by deficient cytochrome c oxidase (complex IV) activity with decreased cytochromes and reduced oxidative phosphorylation. There was also severe mitochondrial damage accompanied by decrease of mitochondria, associated with toxic levels of propionyl CoA as shown by carnitine supplementation studies, which explains the apparently paradoxical mitochondrial diminution in the face of the energy sensor AMPK activation, known to induce mitochondria biogenesis. This idea was supported by experiments on AMPK knockout mouse embryonic fibroblasts (MEFs). The multifactorial ATP deficit also provides a plausible basis for the cardiomyopathy in patients with propionic acidemia, and other diseases.Additionally, systemic inflammation concomitant to the toxic state might explain our findings of enhanced IL-6, STAT3 and HIF-1α, associated with an increase of mitophagic BNIP3 and PINK proteins, which may further increase mitophagy. Together our results imply core mechanisms of energy deficit in several inherited metabolic disorders.

Thiophenecarboxamide Derivatives Activated by EthA Kill Mycobacterium tuberculosis by Inhibiting the CTP Synthetase PyrG.

To combat the emergence of drug-resistant strains of Mycobacterium tuberculosis, new antitubercular agents and novel drug targets are needed. Phenotypic screening of a library of 594 hit compounds uncovered two leads that were active against M. tuberculosis in its replicating, non-replicating, and intracellular states: compounds 7947882 (5-methyl-N-(4-nitrophenyl)thiophene-2-carboxamide) and 7904688 (3-phenyl-N-[(4-piperidin-1-ylphenyl)carbamothioyl]propanamide). Mutants resistant to both compounds harbored mutations in ethA (rv3854c), the gene encoding the monooxygenase EthA, and/or in pyrG (rv1699) coding for the CTP synthetase, PyrG. Biochemical investigations demonstrated that EthA is responsible for the activation of the compounds, and by mass spectrometry we identified the active metabolite of 7947882, which directly inhibits PyrG activity. Metabolomic studies revealed that pharmacological inhibition of PyrG strongly perturbs DNA and RNA biosynthesis, and other metabolic processes requiring nucleotides. Finally, the crystal structure of PyrG was solved, paving the way for rational drug design with this newly validated drug target.

Differential gene expression in the liver of the African lungfish, Protopterus annectens, after 6 months of aestivation in air or 1 day of arousal from 6 months of aestivation.

The African lungfish, Protopterus annectens, can undergo aestivation during drought. Aestivation has three phases: induction, maintenance and arousal. The objective of this study was to examine the differential gene expression in the liver of P. annectens after 6 months (the maintenance phase) of aestivation as compared with the freshwater control, or after 1 day of arousal from 6 months aestivation as compared with 6 months of aestivation using suppression subtractive hybridization. During the maintenance phase of aestivation, the mRNA expression of argininosuccinate synthetase 1 and carbamoyl phosphate synthetase III were up-regulated, indicating an increase in the ornithine-urea cycle capacity to detoxify ammonia to urea. There was also an increase in the expression of betaine homocysteine-S-transferase 1 which could reduce and prevent the accumulation of hepatic homocysteine. On the other hand, the down-regulation of superoxide dismutase 1 expression could signify a decrease in ROS production during the maintenance phase of aestivation. In addition, the maintenance phase was marked by decreases in expressions of genes related to blood coagulation, complement fixation and iron and copper metabolism, which could be strategies used to prevent thrombosis and to conserve energy. Unlike the maintenance phase of aestivation, there were increases in expressions of genes related to nitrogen, carbohydrate and lipid metabolism and fatty acid transport after 1 day of arousal from 6 months aestivation. There were also up-regulation in expressions of genes that were involved in the electron transport system and ATP synthesis, indicating a greater demand for metabolic energy during arousal. Overall, our results signify the importance of sustaining a low rate of waste production and conservation of energy store during the maintenance phase, and the dependence on internal energy store for repair and structural modification during the arousal phase, of aestivation in the liver of P. annectens.

Proximity biotinylation and affinity purification are complementary approaches for the interactome mapping of chromatin-associated protein complexes.

Mapping protein-protein interactions for chromatin-associated proteins remains challenging. Here we explore the use of BioID, a proximity biotinylation approach in which a mutated biotin ligase (BirA*) is fused to a bait of interest, allowing for the local activation of biotin and subsequent biotinylation of proteins in the bait vicinity. BioID allowed for successful interactome mapping of core histones and members of the mediator complex. We explored the background signal produced by the BioID approach and found that using distinct types of controls increased the stringency of our statistical analysis with SAINTexpress. A direct comparison of BioID with our AP-MS protocol optimized for chromatin-associated protein complexes revealed that the approaches identified few shared interaction partners and enriched for distinct biological processes; yet, both approaches permitted the recovery of biologically meaningful interactions. While no clear bias could be observed for either technique toward protein complexes of particular functions, BioID allowed for the purification of proteins of lower cellular abundance. Finally, we were able to identify a strong association of MED4 with the centrosome by BioID and validated this finding by immunofluorescence. In summary, BioID complements AP-MS for the study of chromatin-associated protein complexes. BIOLOGICAL SIGNIFICANCE: This manuscript describes the application of BioID, a proximity biotinylation approach, to chromatin-associated proteins, namely core histones and members of the mediator complex. We observed that BioID was successful at identifying known interaction partners for the baits tested, but also allowed novel putative interaction partners to be identified. By performing a detailed comparison of BioID versus a standard method for interactome mapping (affinity purification coupled to mass spectrometry, AP-MS), we show that the approaches were complementary, allowing for purification of different interaction partners. These interaction partners were different in the biological processes they are associated with, but also in their abundance. BioID represents a significant technical development in the field of chromatin research by expanding the search space for interactome mapping beyond what is possible with AP-MS. This article is part of a Special Issue entitled: Protein dynamics in health and disease. Guest Editors: Pierre Thibault and Anne-Claude Gingras.

Glutamine deprivation initiates reversible assembly of mammalian rods and rings.

Rods and rings (RR) are protein assemblies composed of cytidine triphosphate synthetase type 1 (CTPS1) and inosine monophosphate dehydrogenase type 2 (IMPDH2), key enzymes in CTP and GTP biosynthesis. Small-molecule inhibitors of CTPS1 or IMPDH2 induce RR assembly in various cancer cell lines within 15 min to hours. Since glutamine is an essential amide nitrogen donor in these nucleotide biosynthetic pathways, glutamine deprivation was examined to determine whether it leads to RR formation. HeLa cells cultured in normal conditions did not show RR, but after culturing in media lacking glutamine, short rods (<2 µm) assembled after 24 h, and longer rods (>5 µm) formed after 48 h. Upon supplementation with glutamine or guanosine, these RR underwent almost complete disassembly within 15 min. Inhibition of glutamine synthetase with methionine sulfoximine also increased RR assembly in cells deprived of glutamine. Taken together, our data support the hypothesis that CTP/GTP biosynthetic enzymes polymerize to form RR in response to a decreased intracellular level of glutamine. We speculate that rod and ring formation is an adaptive metabolic response linked to disruption of glutamine homeostasis.

Epigenetic synergies between biotin and folate in the regulation of pro-inflammatory cytokines and repeats.

The protein biotin ligase, holocarboxylase synthetase (HLCS), is a chromatin protein that interacts physically with the DNA methyltransferase DNMT1, the methylated cytosine-binding protein MeCP2 and the histone H3 K9-methyltransferase EHMT1, all of which participate in folate-dependent gene repression. Here we tested the hypothesis that biotin and folate synergize in the repression of pro-inflammatory cytokines and long-terminal repeats (LTRs), mediated by interactions between HLCS and other chromatin proteins. Biotin and folate supplementation could compensate for each other's deficiency in the repression of LTRs in Jurkat and U937 cells. For example, when biotin-deficient Jurkat cells were supplemented with folate, the expression of LTRs decreased by >70%. Epigenetic synergies were more complex in the regulation of cytokines compared with LTRs. For example, the abundance of TNF-α was 100% greater in folate- and biotin-supplemented U937 cells compared with biotin-deficient and folate-supplemented cells. The NF-κB inhibitor curcumin abrogated the effects of folate and biotin in cytokine regulation, suggesting that transcription factor signalling adds an extra layer of complexity to the regulation of cytokine genes by epigenetic phenomena. We conclude that biotin and folate synergize in the repression of LTRs and that these interactions are probably mediated by HLCS-dependent epigenetic mechanisms. In contrast, synergies between biotin and folate in the regulation of cytokines need to be interpreted in the context of transcription factor signalling.

Identification and assessment of markers of biotin status in healthy adults.

Human biotin requirements are unknown and the identification of reliable markers of biotin status is necessary to fill this knowledge gap. Here, we used an outpatient feeding protocol to create states of biotin deficiency, sufficiency and supplementation in sixteen healthy men and women. A total of twenty possible markers of biotin status were assessed, including the abundance of biotinylated carboxylases in lymphocytes, the expression of genes from biotin metabolism and the urinary excretion of biotin and organic acids. Only the abundance of biotinylated 3-methylcrotonyl-CoA carboxylase (holo-MCC) and propionyl-CoA carboxylase (holo-PCC) allowed for distinguishing biotin-deficient and biotin-sufficient individuals. The urinary excretion of biotin reliably identified biotin-supplemented subjects, but did not distinguish between biotin-depleted and biotin-sufficient individuals. The urinary excretion of 3-hydroxyisovaleric acid detected some biotin-deficient subjects, but produced a meaningful number of false-negative results and did not distinguish between biotin-sufficient and biotin-supplemented individuals. None of the other organic acids that were tested were useful markers of biotin status. Likewise, the abundance of mRNA coding for biotin transporters, holocarboxylase synthetase and biotin-dependent carboxylases in lymphocytes were not different among the treatment groups. Generally, datasets were characterised by variations that exceeded those seen in studies in cell cultures. We conclude that holo-MCC and holo-PCC are the most reliable, single markers of biotin status tested in the present study.

Hepatic and extrahepatic distribution of ornithine urea cycle enzymes in holocephalan elephant fish (Callorhinchus milii).

Cartilaginous fish comprise two subclasses, the Holocephali (chimaeras) and Elasmobranchii (sharks, skates and rays). Little is known about osmoregulatory mechanisms in holocephalan fishes except that they conduct urea-based osmoregulation, as in elasmobranchs. In the present study, we examined the ornithine urea cycle (OUC) enzymes that play a role in urea biosynthesis in the holocephalan elephant fish, Callorhinchus milii (cm). We obtained a single mRNA encoding carbamoyl phosphate synthetase III (cmCPSIII) and ornithine transcarbamylase (cmOTC), and two mRNAs encoding glutamine synthetases (cmGSs) and two arginases (cmARGs), respectively. The two cmGSs were structurally and functionally separated into two types: brain/liver/kidney-type cmGS1 and muscle-type cmGS2. Furthermore, two alternatively spliced transcripts with different sizes were found for cmgs1 gene. The longer transcript has a putative mitochondrial targeting signal (MTS) and was predominantly expressed in the liver and kidney. MTS was not found in the short form of cmGS1 and cmGS2. A high mRNA expression and enzyme activities were found in the liver and muscle. Furthermore, in various tissues examined, mRNA levels of all the enzymes except cmCPSIII were significantly increased after hatching. The data show that the liver is the important organ for urea biosynthesis in elephant fish, but, extrahepatic tissues such as the kidney and muscle may also contribute to the urea production. In addition to the role of the extrahepatic tissues and nitrogen metabolism, the molecular and functional characteristics of multiple isoforms of GSs and ARGs are discussed.