Glycinamide Ribonucleotide Transformylase Research Articles

Structures of Glycinamide Ribonucleotide Transformylase (PurN) from Mycobacterium tuberculosis Reveal a Novel Dimer with Relevance to Drug Discovery

Enzymes from the de novo purine biosynthetic pathway have been exploited for the development of anti-cancer drugs, and represent novel targets for anti-bacterial drug development. In Mycobacterium tuberculosis, the cause of tuberculosis, this pathway has been identified as essential for growth and survival. The structure of M. tuberculosis PurN (MtPurN) has been determined in complex with magnesium and iodide at 1.30 Å resolution, and with cofactor analogue, 5-methyltetrahydrofolate (5MTHF) at 2.2 Å resolution. The structure shows a Rossmann-type fold that is very similar to the known structures of the human and E. coli PurN proteins. In contrast, MtPurN forms a dimer that is quite different from that formed by the Escherichia coli PurN, and which suggests a mechanism whereby communication could take place between the two active sites. Differences are seen in two active site loops and in the binding mode of the 5MTHF cofactor analogue between the two MtPurN molecules of the dimer. A binding site for halide ions is found in the dimer interface, and bound magnesium and iodide ions in the active site suggest sites that might be exploited in potential drug discovery strategies.

Mutations in the Chinese hamster ovary cell GART gene of de novo purine synthesis

Mutations in several steps of de novo purine synthesis lead to human inborn errors of metabolism often characterized by mental retardation, hypotonia, sensorineural hearing loss, optic atrophy, and other features. In animals, the phosphoribosylglycinamide transformylase (GART) gene encodes a trifunctional protein carrying out 3 steps of de novo purine synthesis, phosphoribosylglycinamide synthase (GARS), phosphoribosylglycinamide transformylase (also abbreviated as GART), and phosphoribosylaminoimidazole synthetase (AIRS) and a smaller protein that contains only the GARS domain of GART as a functional protein. The GART gene is located on human chromosome 21 and is aberrantly regulated and overexpressed in individuals with Down syndrome (DS), and may be involved in the phenotype of DS. The GART activity of GART requires 10-formyltetrahydrofolate and has been a target for anti-cancer drugs. Thus, a considerable amount of information is available about GART, while less is known about the GARS and AIRS domains. Here we demonstrate that the amino acid residue glu75 is essential for the activity of the GARS enzyme and that the gly684 residue is essential for the activity of the AIRS enzyme by analysis of mutations in the Chinese hamster ovary (CHO-K1) cell that require purines for growth. We report the effects of these mutations on mRNA and protein content for GART and GARS. Further, we discuss the likely mechanisms by which mutations inactivating the GART protein might arise in CHO-K1 cells.

Asymmetric Synthesis of Inhibitors of Glycinamide Ribonucleotide Transformylase

Glycinamide ribonucleotide transformylase (GAR Tfase) catalyzes the first of two formyl transfer steps in the de novo purine biosynthetic pathway that require folate cofactors and has emerged as a productive target for antineoplastic therapeutic intervention. The asymmetric synthesis and evaluation of the two diastereomers of 10-methylthio-DDACTHF (10R-3 and 10S-3) and related analogues as potential inhibitors of GAR Tfase are reported. This work, which defines the importance of the C10 stereochemistry for this class of inhibitors of GAR Tfase, revealed that both diastereomers are potent inhibitors of rhGAR Tfase (10R-3 Ki = 210 nM, 10S-3 Ki = 180 nM) that exhibit effective cell growth inhibition (CCRF-CEM IC50 = 80 and 50 nM, respectively), which is dependent on intracellular polyglutamation by folylpolyglutamate synthetase (FPGS) but not intracellular transport by the reduced folate carrier.

Modeling folate, one-carbon metabolism, and DNA methylation

The B-vitamin folate is essential for one-carbon transfer reactions. These include methylation reactions, such as the methylation of DNA or other substrates (by use of S-adenosylmethionine, SAM, which is concurrently converted to S-adenosylhomocysteine), and also the synthesis of thymidine and purines, which are needed for DNA synthesis and repair (see Figure 1).1 A protective role of folate early in carcinogenesis is supported by epidemiologic observations of inverse associations with the intakes of folate or biomarkers of folate status, by animal experimental studies, and by molecular epidemiologic studies that show associations between folate-related polymorphisms and cancer risk, particularly for colorectal and hematopoietic malignancies.2–4 However, after the establishment of pre-neoplastic lesions, folate may foster carcinogenesis.5,6 Figure 1 Overview of folate-mediated one-carbon metabolism (simplified) links to methylation reactions and nucleotide synthesis. Abbreviations : AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide; AICARFT, 5-amino-imidazole-4-carboxamide ribonucleotide transformylase; DHF, dihydrofolate; DHFR, dihydrofolate reductase; dUMP, deoxyuridine monophosphate; GAR, glycinamide ribonucleotide; GART, glycinamide ribonucleotide transformylase; hFR, human folate receptor; MS, methionine synthase; MT, methyltransferases; MTHFR, 5,10-methylenetetrahydrofolate reductase; RFC, reduced folate carrier; SAH (AdoHcy), S-adenosylhomocysteine; SAM (AdoMet), S-adenosylmethionine; THF, tetrahydrofolate; dTMP, deoxythymidine monophosphate; TS, thymidylate synthase; X, a variety of substrates for methylation. Adapted from Ulrich et al. (2003)35 with permission. The biological mechanisms for the folate-cancer relationship are not yet well defined.1 The link of folate deficiency to reduced provision of nucleotides causing DNA damage subsequently is well established.7 However, the relationship between folate status and epigenetic changes is less clear. DNA methylation disturbances that occur during carcinogenic processes involve both global DNA methylation, largely at repeat or satellite regions of DNA, as well as concurrent promoter-specific …

Glycinamide ribonucleotide transformylase (GAR Tfase) as a target for cancer therapy

Glycinamide ribonucleotide transformylase (GAR Tfase) as a target for cancer therapy

Glycinamide ribonucleotide transformylase (GAR Tfase) as a target for cancer therapy

Glycinamide ribonucleotide transformylase (GAR Tfase) as a target for cancer therapy

Human Glycinamide Ribonucleotide Transformylase: Active Site Mutants as Mechanistic Probes

Human glycinamide ribonucleotide transformylase (GART) (EC 2.1.2.2) is a validated target for cancer chemotherapy, but mechanistic studies of this therapeutically important enzyme are limited. Site-directed mutagenesis, initial velocity studies, pH-rate studies, and substrate binding studies have been employed to probe the role of the strictly conserved active site residues, N106, H108, and D144, and the semiconserved K170 in substrate binding and catalysis. Only two conservative substitutions, N106Q and K170R, resulted in catalytically active enzymes, and these active mutant enzymes gave pH-rate profiles and a steady-state kinetic mechanism essentially identical to those of the native enzyme. All inactive mutants were able to bind both substrates, ruling out disrupted formation of the ternary complex as the source of inactivity. Differences between human and Escherichia coli GART, previously used as a model for the human enzyme, were evident.

The effect of a novel transition state inhibitor of methylthioadenosine phosphorylase on pemetrexed activity

Pemetrexed is a new-generation antifolate inhibitor of thymidylate synthase (TS) and a weaker inhibitor of glycinamide ribonucleotide transformylase (GARFT) required for de novo purine synthesis. Methylthioadenosine phosphorylase (MTAP) salvages purines by releasing adenine from methylthioadenosine and is often deleted in mesothelioma. The current study addresses the effect of MTAP on pemetrexed activity using a highly potent transition state inhibitor of MTAP, MT-DADMe-Immucillin A (ImmA; K(i) = 86 pmol/L) in the MTAP(+) NCI-H28 and MTAP(-) NCI-H2052 mesothelioma cell lines. Based on selective nucleoside protection, TS was found to be the primary pemetrexed target in both cell lines with GARFT inhibition requiring 20- to 30-fold higher pemetrexed concentrations. ImmA had no effect on pemetrexed activity but, when thymidine was added, the pemetrexed IC(50) decreased by a factor of approximately 3 in MTAP(+) H28 cells with no effect in MTAP(-) H2052 cells. Conversely, the transfection of MTAP into H2052 cells increased the pemetrexed IC(50) by nearly 3-fold but only in the presence of thymidine; this was reversed by ImmA. An MTAP-specific short interfering RNA produced a 2-fold decrease in pemetrexed IC(50) in MTAP(+) HeLa cells in the presence of thymidine. These data indicate that suppression of constitutive MTAP has no effect on pemetrexed activity when the primary target is TS. There is a modest salutary effect when the pemetrexed target is GARFT alone.

Molecular mechanisms underlying the enhanced sensitivity of thiopurine-resistant T-lymphoblastic cell lines to methyl mercaptopurineriboside

Methylmercaptopurine riboside (meMPR), a cellular metabolite of 6-mercaptopurine (6-MP), is a potent inhibitor of de novo purine synthesis (DNPS). Human MOLT4 T-lymphoblastic leukaemia cells that have acquired resistance to 6-MP or 6-thioguanine (6-TG) as a consequence of defective transport exhibit enhanced sensitivity to meMPR. HPLC-based analysis of the transport of meMPR revealed normal uptake of this compound by our thiopurine-resistant cell sublines, suggesting a route of transport distinct from that for 6-MP and 6-TG. Studies on the wild-type parental leukemic cells showed that adenosine, dipyridamole and nitrobenzylthioinosine inhibit uptake of meMPR to a significant extent, whereas Na + ions have no influence on this process. Transfection of these leukemic cells with small interference RNA molecules targeting the gene encoding the first member of the family of equiliberative nucleoside transporters (ENT1) strongly reduced the initial rate of meMPR transport. Our resistant cell lines exhibited 30–52% reductions ( p < 0.005) in their levels of mRNA encoding several proteins involved in de novo purine synthesis, i.e., aminoimidazole carboxamide ribonucleotide formyltransferase, glycinamide ribonucleotide transformylase and guanine monophosphate synthetase. Consequently, the rate of de novo purine synthesis in these resistant sublines was decreased by 50%. Furthermore, the levels of ribonucleoside triphosphates in these cells were significantly lower than in the non-resistant parental cells. In combination, a reduced rate of de novo purine synthesis together with low levels of ribonucleoside triphosphates can explain the enhanced sensitivity of our thiopurine-resistant cell lines to meMPR. In this manner, meMPR bypasses the mechanisms of resistance to thiopurines and is even more cytotoxic towards resistant than towards wild-type cells.

Discovery of a Potent, Nonpolyglutamatable Inhibitor of Glycinamide Ribonucleotide Transformylase

Glycinamide ribonucleotide transformylase (GAR Tfase) catalyzes the first of two formyl transfer steps in the de novo purine biosynthetic pathway that require folate cofactors. Herein we report the discovery of a potent, nonpolyglutamatable, and selective inhibitor of GAR Tfase. Compound 12, which possesses a tetrazole in place of the gamma-carboxylic acid in the l-glutamate subunit of the potent GAR Tfase inhibitor 1, was active in cellular-based functional assays exhibiting purine-sensitive cytotoxic activity (IC(50) = 40 nM, CCRF-CEM) and was selective for inhibition of rhGAR Tfase (K(i) = 130 nM). Notably, 12 was only 2.5-fold less potent than 1 in cellular assays and 4-fold less potent against rhGAR Tfase. Like 1, this functional activity of 12 in the cell-based assay benefits from and requires transport into the cell by the reduced folate carrier but, unlike 1, is independent of folyl polyglutamate synthase (FPGS) expression levels and polyglutamation.

The inverse relationship between reduced folate carrier function and Pemetrexed activity in a human colon cancer cell line

Pemetrexed, a new generation antifolate recently approved for the treatment of mesothelioma and non-small cell lung cancer, is an excellent substrate for the reduced folate carrier (RFC). To explore the carrier's effect on pemetrexed activity, RFC was inactivated in HCT-15 colon cancer cells by mutagenesis and PT632 selective pressure. A clone (PT1) was obtained with a glycine to arginine substitution at amino acid 401, resulting in the loss of RFC function. PT1 cells were resistant to PT632 (178-fold), methotrexate (4-fold), and ZD1694 (Tomudex, raltitrexed; 20-fold), but were 3-fold collaterally sensitive to pemetrexed when grown in 25 nmol/L of 5-formyltetrahydrofolate. PT1 cells transfected with wild-type RFC had antifolate sensitivities comparable to that of wild-type HCT-15 cells, indicating that the RFC mutation was the sole basis for resistance. Folate pools were contracted in PT1 cells by 32% or 60%, as measured by radiolabeling intracellular folates or by an enzyme binding assay, respectively. This was reflected in marked (6.5-fold) collateral sensitivity to trimetrexate. The initial uptake of pemetrexed in PT1 cells was markedly reduced ( approximately 85%) but intracellular pemetrexed levels increased to approximately 60% and approximately 70% to that of wild-type cells after 2 hours and 6 days, respectively. There was increased pemetrexed inhibition of glycinamide ribonucleotide transformylase and, to a lesser extent, thymidylate synthase in PT1 cells growing in 5-formyltetrahydrofolate based on nucleoside protection analyses. Hence, loss of RFC function leads to collateral sensitivity to pemetrexed in HCT-15 cells, likely due to cellular folate pool contraction resulting in partial preservation of pemetrexed polyglutamylation and increased target enzyme inhibition. micro

Molecule dynamics and combined QM/MM study on one-carbon unit transfer reaction catalyzed by GAR transformylase

Abstract Both a molecule dynamic study and a combined quantum mechanics and molecule mechanics (QM/MM) study on Glycinamide ribonucleotide transformylase (GAR Tfase) catalytic mechanism are presented. The results indicate a direct one-carbon unit transfer process but not a stepwise mechanism in this reaction. The residues near the active center can fix the cofactor (N10-formyltetrahydrofolate) and GAR in proper relative positions by a H-bond network. The transition state and the minimum energy pathway are located on the potential energy surface. After all the residues (including H2O molecules) are removed from the system the activation energy has increased from 145.1 kJ/mol to 243.3 kJ/mol, and the formly transfer reaction is very hard to achieve. The interactions between coenzyme, GAR and residues near the reactive center are discussed as well.

A quantum chemical study on the mechanism of glycinamide ribonucleotide transformylase inhibitor: 10-Formyl-5,8,10-trideazafolic acid

A density functional theory (DFT) study is presented on the reaction mechanism of glycinamide ribonucleotide (GAR) with 10-formyl-5,8,10-trideazafolic acid (10f-TDAF), which is an inhibitor designed for GAR transformylase (GAR Tfase). There are three different paths for this system and the results indicate that inhibitor 10f-TDAF can form a very stable intermediate with the substrate GAR or generate an imine bond with GAR by elimination of water. The results have verified the presumption from available experiments and implied that 10f-TDAF would be an important target for anti-neoplastic intervention.

The Apo and Ternary Complex Structures of a Chemotherapeutic Target: Human Glycinamide Ribonucleotide Transformylase,

Glycinamide ribonucleotide transformylase (GART; 10-formyltetrahydrofolate:5'-phosphoribosylglycinamide formyltransferase, EC 2.1.2.2), an essential enzyme in de novo purine biosynthesis, has been a chemotherapeutic target for several decades. The three-dimensional structure of the GART domain from the human trifunctional enzyme has been solved by X-ray crystallography. Models of the apoenzyme, and a ternary complex with the 10-formyl-5,8-dideazafolate cosubstrate and a glycinamide ribonucleotide analogue, hydroxyacetamide ribonucleotide [alpha,beta-N-(hydroxyacetyl)-d-ribofuranosylamine], are reported to 2.2 and 2.07 A, respectively. The model of the apoenzyme represents the first structure of GART, from any source, with a completely unoccupied substrate and cosubstrate site, while the ternary complex is the first structure of the human GART domain that is bound at both the substrate and cosubstrate sites. A comparison of the two models therefore reveals subtle structural differences that reflect substrate and cosubstrate binding effects and implies roles for the invariant residues Gly 133, Gly 146, and His 137. Preactivation of the DDF formyl group appears to be key for catalysis, and structural flexibility of the active end of the substrate may facilitate nucleophilic attack. A change in pH, rather than folate binding, correlates with movement of the folate binding loop, whereas the phosphate binding loop position does not vary with pH. The electrostatic surface potentials of the human GART domain and Escherichia coli enzyme explain differences in the binding affinity of polyglutamylated folates, and these differences have implications to future chemotherapeutic agent design.

Structure and Function of Both Domains of ArnA, a Dual Function Decarboxylase and a Formyltransferase, Involved in 4-Amino-4-deoxy-L-arabinose Biosynthesis

Modification of the lipid A moiety of lipopolysaccharide by the addition of the sugar 4-amino-4-deoxy-L-arabinose (L-Ara4N) is a strategy adopted by pathogenic Gram-negative bacteria to evade cationic antimicrobial peptides produced by the innate immune system. L-Ara4N biosynthesis is therefore a potential anti-infective target, because inhibiting its synthesis would render certain pathogens more sensitive to the immune system. The bifunctional enzyme ArnA, which is required for L-Ara4N biosynthesis, catalyzes the NAD(+)-dependent oxidative decarboxylation of UDP-glucuronic acid to generate a UDP-4'-keto-pentose sugar and also catalyzes transfer of a formyl group from N-10-formyltetrahydrofolate to the 4'-amine of UDP-L-Ara4N. We now report the crystal structure of the N-terminal formyltransferase domain in a complex with uridine monophosphate and N-5-formyltetrahydrofolate. Using this structure, we identify the active site of formyltransfer in ArnA, including the key catalytic residues Asn(102), His(104), and Asp(140). Additionally, we have shown that residues Ser(433) and Glu(434) of the decarboxylase domain are required for the oxidative decarboxylation of UDP-GlcUA. An E434Q mutant is inactive, suggesting that chemical rather than steric properties of this residue are crucial in the decarboxylation reaction. Our data suggest that the decarboxylase domain catalyzes both hydride abstraction (oxidation) from the C-4' position and the subsequent decarboxylation.

Synthesis and biological evaluation of α- and γ-carboxamide derivatives of 10-CF 3CO-DDACTHF

Structurally-related, but non-polyglutamylatable, derivatives of 10-CF 3CO-DDACTHF ( 1), which incorporate l-glutamine ( 2) and l-isoglutamine ( 3) in place of l-glutamate, were prepared and evaluated as inhibitors of recombinant human (rh) GAR Tfase. While the l-glutamate α-carboxamide derivative 3 was much less effective as a rhGAR Tfase inhibitor ( K i = 4.8 μM) and inactive in cellular functional assays, the γ-carboxamide derivative 2 was found to be a potent and selective rhGAR Tfase inhibitor ( K i = 0.056 μM) being only 4-fold less potent than 1 ( K i = 0.015 μM). Moreover, 2 was effective in cellular functional assays exhibiting purine sensitive cytotoxic activity (IC 50 = 300 nM, CCRF-CEM) only 20-fold less potent than 1 (IC 50 = 16 nM), consistent with inhibition of de novo purine biosynthesis via selective inhibition of GAR Tfase. Like 1, 2 is transported into the cell by the reduced folate carrier. Unlike 1, the functional activity of 2 is not dependent upon FPGS polyglutamylation.

Design, synthesis, and biological evaluation of 10-methanesulfonyl-DDACTHF, 10-methanesulfonyl-5-DACTHF, and 10-methylthio-DDACTHF as potent inhibitors of GAR Tfase and the de novo purine biosynthetic pathway

The synthesis and evaluation of 10-methanesulfonyl-DDACTHF (1), 10-methanesulfonyl-5-DACTHF (2), and 10-methylthio-DDACTHF (3) as potential inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) are reported. The compounds 10-methanesulfonyl-DDACTHF (1, Ki=0.23μM), 10-methanesulfonyl-5-DACTHF (2, Ki=0.58μM), and 10-methylthio-DDACTHF (3, Ki=0.25μM) were found to be selective and potent inhibitors of recombinant human GAR Tfase. Of these, 3 exhibited exceptionally potent, purine sensitive growth inhibition activity (3, IC50=100nM) against the CCRF–CEM cell line being 3-fold more potent than Lometrexol and 30-fold more potent than the parent, unsubstituted DDACTHF, whereas 1 and 2 exhibited more modest growth inhibition activity (1, IC50=1.0μM and 2, IC50=2.0μM).

Synthesis and biological evaluation of N-{4-[5-(2,4-diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-2-(2,2,2-trifluoroacetyl)pentyl]benzoyl}- l-glutamic acid as a potential inhibitor of GAR Tfase and the de novo purine biosynthetic pathway

The synthesis and evaluation of N-{4-[5-(2,4-diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-2-(2,2,2-trifluoroacetyl)pentyl]benzoyl}- l-glutamic acid ( 2) as an inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) are reported. The inhibitor 2 was prepared in a convergent synthesis involving C-alkylation of methyl 4-(4,4,4-trifluoro-3-dimethylhydrazonobutyl)benzoate with 1-chloro-3-iodopropane followed by construction of the pyrimidinone ring. Compound 2 was found to be an effective inhibitor of recombinant human GAR Tfase ( K i = 0.50 μM), whereas it was inactive ( K i > 100 μM) against E. coli GAR Tfase as well as recombinant human AICAR Tfase. Compound 2 exhibited modest, purine-sensitive growth inhibitory activity against the CCRF-CEM cell line (IC 50 = 6.0 μM).

Alternative splicing of the Vupur3 transcript in cowpea produces multiple mRNA species with a single protein product that is present in both plastids and mitochondria.

A heterogeneous population of cDNAs (designated Vupur3) encoding phosphoribosylglycinamide formyltransferase (GART; EC 2.1.2.2) was isolated from a cowpea (Vigna unguiculata L. Walp.) nodule library. Three classes of cDNA with the same ORF, but differing in their 3'-UTRs, were identified. Southern analysis and sequencing of genomic DNA confirmed that these differences result from alternative splicing of the primary transcript of a single Vupur3 gene. Alternative splicing does not appear to play a role in the production of soybean (Glycine max Merrill.) pur3 transcripts. The presence of the protein product of the Vupur3 gene, GART, in plastids and mitochondria was confirmed by immunoblotting with antibodies raised against the recombinant protein. The antibodies recognised two proteins with apparent molecular masses of 27 and 27.5 kDa in both mitochondria and plastids. All Vupur3 transcripts have two in-frame start codons that are active in wheatgerm in vitro transcription / translation experiments suggesting a mechanism by which the gene product could be targeted to two organelles. Like other genes encoding enzymes for purine synthesis, Vupur3 is expressed in nodules before nitrogen fixation begins but in contrast to these genes its expression does not increase markedly after nitrogen fixation begins.

Mapping of the bovine genes of the de novo AMP synthesis pathway1

Summary The purine nucleotides adenosine monophosphate (AMP) and guanosine monophosphate (GMP) are critical for energy metabolism, cell signalling and cell reproduction. Despite their essential function, little is known about the regulation and in vivo expression pattern of the genes involved in the de novo purine synthesis pathway. The complete coding region of the bovine phosphoribosylaminoimidazole carboxylase gene (PAICS), which catalyses steps 6 and 7 of the de novo purine biosynthesis pathway, as well as bovine genomic sequences of the six other genes in the pathway producing inosine monophosphate (IMP) and AMP [phosphoribosyl pyrophosphate amidotransferase (PPAT), phosphoribosylglycinamide formyltransferase (GART), phosphoribosylformylglycinamidine synthase (PFAS), adenylosuccinate lyase (ADSL), 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC) and adenylosuccinate synthase (ADSS)], were identified. The genes were mapped to segments of six different bovine chromosomes using a radiation hybrid (RH) cell panel. The gene PPAT, coding for the presumed rate-limiting enzyme of the purine de novo pathway was closely linked to PAICS on BTA6. These, and the other bovine locations i.e. GART at BTA1, PFAS at BTA19, ADSL at BTA5, ATIC at BTA2 and ADSS at BTA16, are in agreement with published comparative maps of cattle and man. PAICS and PPAT genes are known to be closely linked in human, rat and chicken. Previously, an expressed sequence fragment of PAICS (Bos taurus corpus luteum, BTCL9) was mapped to BTA13. By isolation and characterization of a BAC clone, we have now identified a PAICS processed pseudogene sequence (psiPAICS) on BTA13. Processed pseudogene sequences of PAICS and other genes of the purine biosynthesis pathway were identified in several mammalian species, indicating that the genes of this pathway have been susceptible to retrotransposition. The seven bovine genes are expressed at a higher level in testicular and ovary tissues compared with skeletal muscle.