Fragment-Based Discovery of MRTX1719, a Synthetic Lethal Inhibitor of the PRMT5•MTA Complex for the Treatment of MTAP-Deleted Cancers.

The PRMT5•MTA complex has recently emerged as a new synthetically lethal drug target for the treatment of MTAP-deleted cancers. Here, we report the discovery of development candidate MRTX1719. MRTX1719 is a potent and selective binder to the PRMT5•MTA complex and selectively inhibits PRMT5 activity in MTAP-deleted cells compared to MTAP-wild-type cells. Daily oral administration of MRTX1719 to tumor xenograft-bearing mice demonstrated dose-dependent inhibition of PRMT5-dependent symmetric dimethylarginine protein modification in MTAP-deleted tumors that correlated with antitumor activity. A 4-(aminomethyl)phthalazin-1(2H)-one hit was identified through a fragment-based screen, followed by X-ray crystallography, to confirm binding to the PRMT5•MTA complex. Fragment growth supported by structural insights from X-ray crystallography coupled with optimization of pharmacokinetic properties aided the discovery of development candidate MRTX1719.

Homozygous MTAP deletion in primary human glioblastoma is not associated with elevation of methylthioadenosine.

Homozygous deletion of methylthioadenosine phosphorylase (MTAP) in cancers such as glioblastoma represents a potentially targetable vulnerability. Homozygous MTAP-deleted cell lines in culture show elevation of MTAP's substrate metabolite, methylthioadenosine (MTA). High levels of MTA inhibit protein arginine methyltransferase 5 (PRMT5), which sensitizes MTAP-deleted cells to PRMT5 and methionine adenosyltransferase 2A (MAT2A) inhibition. While this concept has been extensively corroborated in vitro, the clinical relevance relies on exhibiting significant MTA accumulation in human glioblastoma. In this work, using comprehensive metabolomic profiling, we show that MTA secreted by MTAP-deleted cells in vitro results in high levels of extracellular MTA. We further demonstrate that homozygous MTAP-deleted primary glioblastoma tumors do not significantly accumulate MTA in vivo due to metabolism of MTA by MTAP-expressing stroma. These findings highlight metabolic discrepancies between in vitro models and primary human tumors that must be considered when developing strategies for precision therapies targeting glioblastoma with homozygous MTAP deletion.

NUDT15: A bench to bedside success story.

Recently, the enzyme nudix hydrolase 15 (NUDT15) has been identified as an additional component of the thiopurine metabolism pathway. NUDT15 (also known as MTH2) catalyzes the dephosphorylation of 6-thioguanosine triphosphate (6-TGTP) and 6-thio-deoxyguanosine triphosphate (6-TdGTP), which is the active metabolite of thiopurine medications. Thiopurine compounds, which were first synthesized in the 1950s, are widely used in the treatment of childhood leukemia, inflammatory bowel disease, and autoimmune disorders. For many years, TPMT has been recognized as an enzyme that is involved in thiopurine metabolism, and interindividual variation in TPMT activity has been known to contribute to differences in risk of thiopurine toxicity. Genetic variation that leads to decreased NUDT15 activity has been recognized as an additional contributor, beyond TPMT, to thiopurine toxicity. In some populations, including Asian and Latino populations, NUDT15 genetic variants are more common than TPMT variants, making this a significant biomarker of toxicity. Clinical genetic testing is now available for a subset of NUDT15 variants, representing a remarkably fast translation from bench to bedside. This review will focus on NUDT15 - from discovery to clinical implementation.

Plasmodium falciparum purine nucleoside phosphorylase as a model in the search for new inhibitors by high throughput screening.

Studies have shown that inhibition of Plasmodium falciparum Purine Nucleoside Phosphorylase (PfPNP) blocks the purine salvage pathway in vitro and in vivo. In this study, PfPNP was evaluated as a model in the search for new inhibitors using surface plasmon resonance (SPR). Its expression, purification, oligomeric state, kinetic constants, calorimetric parameters and kinetic mechanisms were obtained. PfPNP was immobilized on a CM5 sensor chip and sensorgrams were produced through binding the enzyme to the substrate MESG and interactions between molecules contained in 10 fractions of natural extracts. The oligomeric state showed that recombinant PfPNP is a hexamer. The true steady-state kinetic parameters for the substrate inosine were: KM 17 µM, kcat 1.2 s-1, VMax 2.2 U/mg and kcat/KM 7 × 10-4; for MESG they were: KM 131 µM, kcat 2.4 s-1, VMax 4.4 U/mg and kcat/KM 1.8 × 10-4. The thermodynamic parameters for the substrate Phosphate were: ΔG - 5.8 cal mol-1, ΔH - 6.5 cal mol-1 and ΔS - 2.25 cal mol-1/degree. The ITC results demonstrated that the binding of phosphate to free PfPNP led to a significant change in heat and association constants and thermodynamic parameters. A sequential ordered mechanism was proposed as the kinetic mechanism. Three plant extracts contained molecules capable of interacting with PfPNP, showing different levels of affinity. The identification of plant extract fractions containing molecules that interact with recombinant PfPNP using SRP validates this target as a model in the search for new inhibitors. In this study, we showed for the first time the true steady-state kinetic parameters for reactions catalyzed by PfPNP and a model using PfPNP as a target for High-throughput Screening for new inhibitors through SPR. This knowledge will allow for the development of more efficient research methods in the search for new drugs against malaria.

Williams-Beuren syndrome-related methyltransferase WBSCR27: cofactor binding and cleavage.

Williams-Beuren syndrome, characterized by numerous physiological and mental problems, is caused by the heterozygous deletion of chromosome region 7q11.23, which results in the disappearance of 26 protein-coding genes. Protein WBSCR27 is a product of one of these genes whose biological function has not yet been established and for which structural information has been absent until now. Using NMR, we investigated the structural and functional properties of murine WBSCR27. For protein in the apo form and in a complex with S-(5'-adenosyl)-l-homocysteine (SAH), a complete NMR resonance assignment has been obtained and the secondary structure has been determined. This information allows us to attribute WBSCR27 to Class I methyltransferases. The interaction of WBSCR27 with the cofactor S-(5'-adenosyl)-l-methionine (SAM) and its metabolic products - SAH, 5'-deoxy-5'-methylthioadenosine (MTA) and 5'-deoxyadenosine (5'dAdo) - was studied by NMR and isothermal titration calorimetry. SAH binds WBSCR27 much tighter than SAM, leaving open the question of cofactor turnover in the methylation reaction. One possible answer to this question is the presence of weak but detectable nucleosidase activity for WBSCR27. We found that the enzyme catalyses the cleavage of the adenine moiety from SAH, MTA and 5'dAdo, similar to the action of bacterial SAH/MTA nucleosidases. We also found that the binding of SAM or SAH causes a significant change in the structure of WBSCR27 and in the conformational mobility of the protein fragments, which can be attributed to the substrate recognition site. This indicates that the binding of the cofactor modulates the folding of the substrate-recognizing region of the enzyme.

Iron deficiency leads to repression of a non-canonical methionine salvage pathway in Schizosaccharomyces pombe.

The methionine salvage pathway (MSP) regenerates methionine from 5'-methylthioadenosine (MTA). Aerobic MSP consists of six enzymatic steps. The mug14+ and adi1+ genes that are involved in the third and fifth steps of the pathway are repressed when Schizosaccharomyces pombe undergoes a transition from high- to low-iron conditions. Results consistently show that methionine auxotrophic cells (met6Δ) require iron for growth in the presence of MTA as the sole source of methionine. Inactivation of the iron-using protein Adi1 leads to defects in the utilization of MTA. In the case of the third step of the pathway, co-expression of two distinct proteins, Mta3 and Mde1, is required. These proteins are interdependent to rescue MTA-dependent growth deficit of met6Δ cells. Coimmunoprecipitation experiments showed that Mta3 is a binding partner of Mde1. Meiotic met6Δ cells co-expressing mta3+ and mde1+ or mta3+ and mug14+ produce comparable levels of spores in the presence of MTA, revealing that Mde1 and Mug14 share a common function when co-expressed with Mta3 in sporulating cells. In sum, our findings unveil several novel features of MSP, especially with respect to its regulation by iron and the discovery of a non-canonical third enzymatic step in the fission yeast.

A bifunctional salvage pathway for two distinct S-adenosylmethionine by-products that is widespread in bacteria, including pathogenic Escherichia coli.

S-adenosyl-l-methionine (SAM) is a necessary cosubstrate for numerous essential enzymatic reactions including protein and nucleotide methylations, secondary metabolite synthesis and radical-mediated processes. Radical SAM enzymes produce 5'-deoxyadenosine, and SAM-dependent enzymes for polyamine, neurotransmitter and quorum sensing compound synthesis produce 5'-methylthioadenosine as by-products. Both are inhibitory and must be addressed by all cells. This work establishes a bifunctional oxygen-independent salvage pathway for 5'-deoxyadenosine and 5'-methylthioadenosine in both Rhodospirillum rubrum and Extraintestinal Pathogenic Escherichia coli. Homologous genes for this pathway are widespread in bacteria, notably pathogenic strains within several families. A phosphorylase (Rhodospirillum rubrum) or separate nucleoside and kinase (Escherichia coli) followed by an isomerase and aldolase sequentially function to salvage these two wasteful and inhibitory compounds into adenine, dihydroxyacetone phosphate and acetaldehyde or (2-methylthio)acetaldehyde during both aerobic and anaerobic growth. Both SAM by-products are metabolized with equal affinity during aerobic and anaerobic growth conditions, suggesting that the dual-purpose salvage pathway plays a central role in numerous environments, notably the human body during infection. Our newly discovered bifunctional oxygen-independent pathway, widespread in bacteria, salvages at least two by-products of SAM-dependent enzymes for carbon and sulfur salvage, contributing to cell growth.

Selective PRMT5 Inhibitors Suppress Human CD8+ T Cells by Upregulation of p53 and Impairment of the AKT Pathway Similar to the Tumor Metabolite MTA.

Genetic alterations in tumor cells provide promising targets for antitumor therapy. Recently, loss of methylthioadenosine phosphorylase (MTAP), a deletion frequently occurring in cancer, has been shown to create vulnerability to the inhibition of the protein arginine methyltransferase 5 (PRMT5). MTAP deficiency leads to accumulation of methylthioadenosine (MTA), which reduces PRMT5 activity, and thus, sensitizes the tumor cells to selective PRMT5 inhibitors (PRMT5i). PRMT5i are investigated as a new strategy to selectively kill MTAP-deficient tumor cells by blocking residual PRMT5 activity, but also to treat PRMT5-overexpressing tumors. Although many studies investigated the role of PRMT5 in cancer, only little data exist about the effect of PRMT5 inhibition on immune cells. As we could show that the tumor metabolite MTA suppresses T cells, we asked whether selective PRMT5 inhibition is detrimental for T-cell immune responses. Therefore, we examined the effect of the synthetic PRMT5 inhibitor EPZ015666 on human CD8+ T cells in direct comparison with the naturally occurring PRMT5-inhibiting molecule MTA. Both compounds reduced T-cell proliferation, viability, and functionality. In addition, T-cell metabolism was impaired upon PRMT5 inhibition. These effects coincided with the induction of p53 expression and reduced AKT/mTOR signaling. Our data clearly demonstrate that PRMT5 activity is involved in various cellular processes of human CD8+ T cells associated with essential T-cell functions. Therefore, not only tumor cells, but also antitumor immune responses, are compromised by PRMT5 inhibitors. This emphasizes the importance of considering side effects on the immune system when developing new strategies to specifically target not only MTAP-deficient tumors.

Characterization of 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidases from Borrelia burgdorferi: Antibiotic targets for Lyme disease.

BACKGROUND: Borrelia burgdorferi causes Lyme disease, the most common tick-borne illness in the United States. The Center for Disease Control and Prevention estimates that the occurrence of Lyme disease in the U.S. has now reached approximately 300,000 cases annually. Early stage Borrelia burgdorferi infections are generally treatable with oral antibiotics, but late stage disease is more difficult to treat and more likely to lead to post-treatment Lyme disease syndrome. METHODS: Here we examine three unique 5'-methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidases (MTNs or MTANs, EC 3.2.2.9) responsible for salvage of adenine and methionine in B. burgdorferi and explore their potential as antibiotic targets to treat Lyme disease. Recombinant Borrelia MTNs were expressed and purified from E. coli. The enzymes were extensively characterized for activity, specificity, and inhibition using a UV spectrophotometric assay. In vitro antibiotic activities of MTN inhibitors were assessed using a bioluminescent BacTiter-Glo™ assay. RESULTS: The three Borrelia MTNs showed unique activities against the native substrates MTA, SAH, and 5'-deoxyadenosine. Analysis of substrate analogs revealed that specific activity rapidly dropped as the length of the 5'-alkylthio substitution increased. Non-hydrolysable nucleoside transition state analogs demonstrated sub-nanomolar enzyme inhibition constants. Lastly, two late stage transition state analogs exerted in vitro IC50 values of 0.3-0.4 µg/mL against cultured B. burgdorferi cells. CONCLUSION: B. burgdorferi is unusual in that it expresses three distinct MTNs (cytoplasmic, membrane bound, and secreted) that are effectively inactivated by nucleoside analogs. GENERAL SIGNIFICANCE: The Borrelia MTNs appear to be promising targets for developing new antibiotics to treat Lyme disease.

Phosphorothioation of foreign DNA influences the transformation efficiency in Clostridium perfringens NCTC8239.

Major advances in Clostridium perfringens genetics have been achieved through the development of electroporation-induced transformation; however, highly transformable strains are still limited. SM101 is the only useful strain for genetic manipulation via transformation in C. perfringens causing foodborne illness (FBI). We focused on the FBI strain NCTC8239, which is transformed at a low frequency, because it has a gene cassette that is predicted to encode enzymes involved in DNA phosphorothioation (PT). The oxidant-dependent degradation of NCTC8239 genomic DNA suggested that the genome is PT-modified. When foreign DNA was PT-modified using a plasmid expressing Salmonella enterica PT modification enzymes, the transformation efficiency of NCTC8239 was significantly higher than that using an unmodified plasmid. We then attempted to establish a highly transformable derivative of NCTC8239, and focused on DptFGH, which are predicted to be PT restriction enzymes. A dptG-null mutant exhibited significantly higher transformation efficiency with unmodified foreign DNA than did the wild-type strain. Furthermore, the mutant was transformed with the unmodified plasmid as efficiently as with a PT-modified plasmid, implying that DptG is involved in PT-dependent restriction. Thus, the present results revealed that PT modifications of foreign DNA influence the transformation frequency of NCTC8239 and suggest that PT is a factor contributing to transformation efficiency in NCTC8239.

The Crystal Structure of Dph2 in Complex with Elongation Factor 2 Reveals the Structural Basis for the First Step of Diphthamide Biosynthesis.

Elongation factor 2 (EF-2), a five-domain, GTP-dependent ribosomal translocase of archaebacteria and eukaryotes, undergoes post-translational modification to form diphthamide on a specific histidine residue in domain IV prior to binding the ribosome. The first step of diphthamide biosynthesis in archaebacteria is catalyzed by Dph2, a homodimeric radical S-adenosylmethionine (SAM) enzyme having a noncanonical architecture. Here, we describe a 3.5 Å resolution crystal structure of the Methanobrevibacter smithii (Ms) Dph2 homodimer bound to two molecules of MsEF-2, one of which is ordered and the other largely disordered. MsEF-2 is bound to both protomers of MsDph2, with domain IV bound to the active site of one protomer and domain III bound to a surface α-helix of an adjacent protomer. The histidine substrate of domain IV is inserted into the active site, which reveals for the first time the architecture of the Dph2 active site in complex with its target substrate. We also determined a high-resolution crystal structure of isolated MsDph2 bound to 5'-methylthioadenosine that shows a conserved arginine residue preoriented by conserved phenylalanine and aspartate residues for binding the carboxylate group of SAM. Mutagenesis experiments suggest that the arginine plays an important role in the first step of diphthamide biosynthesis.

4'-[methyl-11C]-thiothymidine as a proliferation imaging tracer for detection of colorectal cancer: comparison with 18F-FDG.

OBJECTIVE: The novel radiotracer, 4'-[methyl-11C]-thiothymidine (11C-4DST), was developed based on the DNA incorporation method as a cell proliferation marker. This study investigated the feasibility of 11C-4DST positron emission tomography/computed tomography (PET/CT) for detection of colorectal cancer, as compared with 2-deoxy-2-18F-fluoro-D-glucose (18F-FDG) PET/CT, and to correlate the two radiotracers with proliferative activity. METHODS: A total of 18 patients with newly diagnosed colorectal cancer underwent both 11C-4DST and 18F-FDG PET/CT. Tumor lesions were identified as areas of focally increased uptake, exceeding that of adjacent normal tissue. For semiquantitative analysis, the maximal standardized uptake value (SUVmax) was calculated. Proliferative activity as quantified by the Ki-67 index was estimated in tumor specimens. RESULTS: In all 18 patients, colorectal cancers were detected by both 11C-4DST and 18F-FDG PET/CT. The median (± SD) SUVmax for 11C-4DST (6.02 ± 2.55) was significantly lower than that for 18F-FDG (13.91 ± 7.62) (P < 0.001). 11C-4DST SUVmax and 18F-FDG SUVmax showed a significant correlation (r = 0.69, P = 0.002). 11C-4DST SUVmax and Ki-67 index were weakly correlated (r = 0.50, P = 0.04). 18F-FDG SUVmax and Ki-67 index were not significantly correlated (r = 0.44, P = 0.06). CONCLUSIONS: Despite a significantly lower uptake of 11C-4DST than that of 18F-FDG, detection of colorectal cancer was also feasible with 11C-4DST PET/CT. 11C-4DST PET/CT might have a role in the noninvasive assessment of proliferation in colorectal cancer.

The methionine salvage pathway-involving ADI1 inhibits hepatoma growth by epigenetically altering genes expression via elevating S-adenosylmethionine.

The 5'-methylthioadenosine (MTA) cycle-participating human acireductone dioxygenase 1 (ADI1) has been implicated as a tumor suppressor in prostate cancer, yet its role remains unclear in hepatocellular carcinoma (HCC). Here, we demonstrated a significant reduction of ADI1, either in protein or mRNA level, in HCC tissues. Additionally, higher ADI1 levels were associated with favorable postoperative recurrence-free survival in HCC patients. By altering ADI1 expression in HCC cells, a negative correlation between ADI1 and cell proliferation was observed. Cell-based and xenograft experiments were performed by using cells overexpressing ADI1 mutants carrying mutations at the metal-binding sites (E94A and H133A, respectively), which selectively disrupted differential catalytic steps, resulting in staying or leaving the MTA cycle. The results showed that the growth suppression effect was mediated by accelerating the MTA cycle. A cDNA microarray analysis followed by verification experiments identified that caveolin-1 (CAV1), a growth-promoting protein in HCC, was markedly decreased upon ADI1 overexpression. Suppression of CAV1 expression was mediated by an increase of S-adenosylmethionine (SAMe) level. The methylation status of CAV1 promoter was significantly altered upon ADI1 overexpression. Finally, a genome-wide methylation analysis revealed that ADI1 overexpression altered promoter methylation profiles in a set of cancer-related genes, including CAV1 and genes encoding antisense non-coding RNAs, long non-coding RNAs, and microRNAs, resulting in significant changes of their expression levels. In conclusion, ADI1 expression promoted MTA cycle to increase SAMe levels, which altered genome-wide promoter methylation profiles, resulting in altered gene expression and HCC growth suppression.

Expanding the Nucleoside Recoding Toolkit: Revealing RNA Population Dynamics with 6-Thioguanosine.

RNA-sequencing (RNA-seq) measures RNA abundance in a biological sample but does not provide temporal information about the sequenced RNAs. Metabolic labeling can be used to distinguish newly made RNAs from pre-existing RNAs. Mutations induced from chemical recoding of the hydrogen bonding pattern of the metabolic label can reveal which RNAs are new in the context of a sequencing experiment. These nucleotide recoding strategies have been developed for a single uridine analogue, 4-thiouridine (s4U), limiting the scope of these experiments. Here we report the first use of nucleoside recoding with a guanosine analogue, 6-thioguanosine (s6G). Using TimeLapse sequencing (TimeLapse-seq), s6G can be recoded under RNA-friendly oxidative nucleophilic-aromatic substitution conditions to produce adenine analogues (substituted 2-aminoadenosines). We demonstrate the first use of s6G recoding experiments to reveal transcriptome-wide RNA population dynamics.

Correlation of 4'-[methyl-11C]-thiothymidine uptake with human equilibrative nucleoside transporter-1 and thymidine kinase-1 expressions in patients with newly diagnosed gliomas.

OBJECTIVE: We examined expressions of human equilibrative nucleoside transporter-1 (hENT1) and thymidine kinase-1 (TK1), the key enzyme in 4'-[methyl-11C]-thiothymidine (4DST) phosphorylation, to elucidate the mechanism of 4DST uptake in patients with newly diagnosed gliomas. METHODS: A total of 19 patients with newly diagnosed gliomas were examined with 4DST PET. Tumor lesions were identified as areas of focally increased uptake, exceeding that of normal brain background. For semi-quantitative analysis, tumor-to-contralateral normal brain tissue (T/N) ratio was determined by dividing the maximal standardized uptake value (SUV) for tumor by that of the mean SUV for reference tissue. The expressions of hENT1, TK1 and Ki-67 in tumor specimens were examined by immunohistochemistry and compared with 4DST T/N ratio. RESULTS: All but two gliomas showed focally increased 4DST uptake. All gliomas showed hENT1 staining, except one grade II glioma, which was also not visualized on 4DST PET. A significant correlation was observed between T/N ratio and hENT1 score (ρ = 0.90, p < 0.001). All gliomas showed TK1 staining, except two gliomas which were also not visualized on 4DST PET. There was a significant correlation between T/N ratio and TK1 score (ρ = 0.92, p < 0.001). There was a significant correlation between T/N ratio and Ki-67 index (ρ = 0.50, p < 0.03). CONCLUSION: Results of this preliminary study indicate that expressions of hENT1 and TK1 appear to be important determinants of 4DST uptake in newly diagnosed gliomas.

Two Distinct Aerobic Methionine Salvage Pathways Generate Volatile Methanethiol in Rhodopseudomonas palustris.

5'-Methyl-thioadenosine (MTA) is a dead-end, sulfur-containing metabolite and cellular inhibitor that arises from S-adenosyl-l-methionine-dependent reactions. Recent studies have indicated that there are diverse bacterial methionine salvage pathways (MSPs) for MTA detoxification and sulfur salvage. Here, via a combination of gene deletions and directed metabolite detection studies, we report that under aerobic conditions the facultatively anaerobic bacterium Rhodopseudomonas palustris employs both an MTA-isoprenoid shunt identical to that previously described in Rhodospirillum rubrum and a second novel MSP, both of which generate a methanethiol intermediate. The additional R. palustris aerobic MSP, a dihydroxyacetone phosphate (DHAP)-methanethiol shunt, initially converts MTA to 2-(methylthio)ethanol and DHAP. This is identical to the initial steps of the recently reported anaerobic ethylene-forming MSP, the DHAP-ethylene shunt. The aerobic DHAP-methanethiol shunt then further metabolizes 2-(methylthio)ethanol to methanethiol, which can be directly utilized by O-acetyl-l-homoserine sulfhydrylase to regenerate methionine. This is in contrast to the anaerobic DHAP-ethylene shunt, which metabolizes 2-(methylthio)ethanol to ethylene and an unknown organo-sulfur intermediate, revealing functional diversity in MSPs utilizing a 2-(methylthio)ethanol intermediate. When MTA was fed to aerobically growing cells, the rate of volatile methanethiol release was constant irrespective of the presence of sulfate, suggesting a general housekeeping function for these MSPs up through the methanethiol production step. Methanethiol and dimethyl sulfide (DMS), two of the most important compounds of the global sulfur cycle, appear to arise not only from marine ecosystems but from terrestrial ones as well. These results reveal a possible route by which methanethiol might be biologically produced in soil and freshwater environments.IMPORTANCE Biologically available sulfur is often limiting in the environment. Therefore, many organisms have developed methionine salvage pathways (MSPs) to recycle sulfur-containing by-products back into the amino acid methionine. The metabolically versatile bacterium Rhodopseudomonas palustris is unusual in that it possesses two RuBisCOs and two RuBisCO-like proteins. While RuBisCO primarily serves as the carbon fixation enzyme of the Calvin cycle, RuBisCOs and certain RuBisCO-like proteins have also been shown to function in methionine salvage. This work establishes that only one of the R. palustris RuBisCO-like proteins functions as part of an MSP. Moreover, in the presence of oxygen, to salvage sulfur, R. palustris employs two pathways, both of which result in production of volatile methanethiol, a key compound of the global sulfur cycle. When total available sulfur was plentiful, methanethiol was readily released into the environment. However, when sulfur became limiting, methanethiol release decreased, presumably due to methanethiol utilization to regenerate needed methionine.

Leishmania infantum 5'-Methylthioadenosine Phosphorylase presents relevant structural divergence to constitute a potential drug target.

BACKGROUND: The 5'-methylthioadenosine phosphorylase (MTAP), an enzyme involved in purine and polyamine metabolism and in the methionine salvage pathway, is considered as a potential drug target against cancer and trypanosomiasis. In fact, Trypanosoma and Leishmania parasites lack de novo purine pathways and rely on purine salvage pathways to meet their requirements. Herein, we propose the first comprehensive bioinformatic and structural characterization of the putative Leishmania infantum MTAP (LiMTAP), using a comparative computational approach. RESULTS: Sequence analysis showed that LiMTAP shared higher identity rates with the Trypanosoma brucei (TbMTAP) and the human (huMTAP) homologs as compared to the human purine nucleoside phosphorylase (huPNP). Motifs search using MEME identified more common patterns and higher relatedness of the parasite proteins to the huMTAP than to the huPNP. The 3D structures of LiMTAP and TbMTAP were predicted by homology modeling and compared to the crystal structure of the huMTAP. These models presented conserved secondary structures compared to the huMTAP, with a similar topology corresponding to the Rossmann fold. This confirmed that both LiMTAP and TbMTAP are members of the NP-I family. In comparison to the huMTAP, the 3D model of LiMTAP showed an additional α-helix, at the C terminal extremity. One peptide located in this specific region was used to generate a specific antibody to LiMTAP. In comparison with the active site (AS) of huMTAP, the parasite ASs presented significant differences in the shape and the electrostatic potentials (EPs). Molecular docking of 5'-methylthioadenosine (MTA) and 5'-hydroxyethylthio-adenosine (HETA) on the ASs on the three proteins predicted differential binding modes and interactions when comparing the parasite proteins to the human orthologue. CONCLUSIONS: This study highlighted significant structural peculiarities, corresponding to functionally relevant sequence divergence in LiMTAP, making of it a potential drug target against Leishmania.

Microbial pathway for anaerobic 5'-methylthioadenosine metabolism coupled to ethylene formation.

Numerous cellular processes involving S-adenosyl-l-methionine result in the formation of the toxic by-product, 5'-methylthioadenosine (MTA). To prevent inhibitory MTA accumulation and retain biologically available sulfur, most organisms possess the "universal" methionine salvage pathway (MSP). However, the universal MSP is inherently aerobic due to a requirement of molecular oxygen for one of the key enzymes. Here, we report the presence of an exclusively anaerobic MSP that couples MTA metabolism to ethylene formation in the phototrophic bacteria Rhodospirillum rubrum and Rhodopseudomonas palustris In vivo metabolite analysis of gene deletion strains demonstrated that this anaerobic MSP functions via sequential action of MTA phosphorylase (MtnP), 5-(methylthio)ribose-1-phosphate isomerase (MtnA), and an annotated class II aldolase-like protein (Ald2) to form 2-(methylthio)acetaldehyde as an intermediate. 2-(Methylthio)acetaldehyde is reduced to 2-(methylthio)ethanol, which is further metabolized as a usable organic sulfur source, generating stoichiometric amounts of ethylene in the process. Ethylene induction experiments using 2-(methylthio)ethanol versus sulfate as sulfur sources further indicate anaerobic ethylene production from 2-(methylthio)ethanol requires protein synthesis and that this process is regulated. Finally, phylogenetic analysis reveals that the genes corresponding to these enzymes, and presumably the pathway, are widespread among anaerobic and facultatively anaerobic bacteria from soil and freshwater environments. These results not only establish the existence of a functional, exclusively anaerobic MSP, but they also suggest a possible route by which ethylene is produced by microbes in anoxic environments.

Structural and Functional Analyses of Periplasmic 5'-Methylthioadenosine/S-Adenosylhomocysteine Nucleosidase from Aeromonas hydrophila.

The Gram-negative, rod-shaped bacterium Aeromonas hydrophila has two multifunctional 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) enzymes, MtaN-1 and MtaN-2, that differ from those in other bacteria. These proteins are essential for several metabolic pathways, including biological methylation, polyamine biosynthesis, methionine recycling, and bacterial quorum sensing. To gain insight into how these two proteins function, we determined four high-resolution crystal structures of MtaN-1 in its apo form and in complex with the substrates S-adenosyl-l-homocysteine, 5'-methylthioadenosine, and 5'-deoxyadenosine. We found that the domain structures were generally similar, although slight differences were evident. The crystal structure demonstrates that AhMtaN-1 has an extension of the binding pocket and revealed that a tryptophan in the active site (Trp199) may play a major role in substrate binding, unlike in other MTAN proteins. Mutation of the Trp199 residue completely abolished the enzyme activity. Trp199 was identified as an active site residue that is essential for catalysis. Furthermore, biochemical characterization of AhMtaN-1 and AhMtaN-2 demonstrated that AhMtaN-1 exhibits inherent trypsin resistance that is higher than that of AhMtaN-2. Additionally, the thermally unfolded AhMtaN-2 protein is capable of refolding into active forms, whereas the thermally unfolded AhMtaN-1 protein does not have this ability. Examining the different biochemical characteristics related to the functional roles of AhMtaN-1 and AhMtaN-2 would be interesting. Indeed, the biochemical characterization of these structural features would provide a structural basis for the design of new antibiotics against A. hydrophila.