Testosterone and persistent organic pollutants in East Greenland male polar bears (Ursus maritimus).

Legacy persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs) are chemicals that undergo long-range transport to the Arctic. These chemicals possess endocrine disruptive properties raising concerns for development and reproduction. Here, we report the relationship between concentrations of testosterone (T) and persistent organic pollutant (POPs) in 40 East Greenland male polar bears (Ursus maritimus) sampled during January to September 1999-2001. The mean ± standard concentrations of blood T were 0.31 ± 0.49 (mean ± SD) ng/mL in juveniles/subadults (n = 22) and 3.58 ± 7.45 ng/mL in adults (n = 18). The ∑POP concentrations (mean ± SD) in adipose tissue were 8139 ± 2990 ng/g lipid weight (lw) in juveniles/subadults and 11,037 ± 3950 ng/g lw in adult males, respectively, of which Σpolychlorinated biphenyls (ΣPCBs) were found in highest concentrations. The variation in T concentrations explained by sampling date (season), biometrics and adipose tissue POP concentrations was explored using redundancy analysis (RDA). The results showed that age, body length, and adipose lipid content in adult males contributed (p = 0.02) to the variation in POP concentrations. However, although some significant relationships between individual organochlorine contaminants and T concentrations in both juveniles/subadults and adult polar bears were identified, no significant relationships (p = 0.32) between T and POP concentrations were identified by the RDAs. Our results suggest that confounders such as biometrics and reproductive status may mask the endocrine disruptive effects that POPs have on blood T levels in male polar bears, demonstrating why it can be difficult to detect effects on wildlife populations.

Ornithine aminotransferase supports polyamine synthesis in pancreatic cancer.

There is a need to develop effective therapies for pancreatic ductal adenocarcinoma (PDA), a highly lethal malignancy with increasing incidence1 and poor prognosis2. Although targeting tumour metabolism has been the focus of intense investigation for more than a decade, tumour metabolic plasticity and high risk of toxicity have limited this anticancer strategy3,4. Here we use genetic and pharmacological approaches in human and mouse in vitro and in vivo models to show that PDA has a distinct dependence on de novo ornithine synthesis from glutamine. We find that this process, which is mediated through ornithine aminotransferase (OAT), supports polyamine synthesis and is required for tumour growth. This directional OAT activity is usually largely restricted to infancy and contrasts with the reliance of most adult normal tissues and other cancer types on arginine-derived ornithine for polyamine synthesis5,6. This dependency associates with arginine depletion in the PDA tumour microenvironment and is driven by mutant KRAS. Activated KRAS induces the expression of OAT and polyamine synthesis enzymes, leading to alterations in the transcriptome and open chromatin landscape in PDA tumour cells. The distinct dependence of PDA, but not normal tissue, on OAT-mediated de novo ornithine synthesis provides an attractive therapeutic window for treating patients with pancreatic cancer with minimal toxicity.

Spatiotemporal water quality variability in a highly loaded surface flow wastewater treatment wetland.

This study evaluates spatiotemporal relationships between water quality parameters (WQPs), nutrients, suspended solids, and biochemical oxygen demand (BOD) concentrations within an engineered wastewater treatment wetland system in the Georgia Piedmont, USA. We explored factors related to treatment efficiency within a heavily loaded 630-m2 surface flow wetland system over a 2-yr period. Relationships between temperature, dissolved oxygen (DO), and oxidation-reduction potential (ORP) were observed; relationships were also seen between these WQPs and nutrient concentrations. Because temperature, DO, and ORP affect nitrogen (N) cycling rates, seasonal trends in N forms were evident in the system. Organic N and inorganic/organic phosphorus concentrations correlated with solids concentrations in the vegetated system without exhibiting seasonal trends. Surface water within the vegetated section generally exhibited anoxic conditions, leading to removal of nitrate-N within the system; however, limited mineralization and nitrification occurred, which greatly limited overall N removal. Plant selection and lack of maintenance likely led to high solids and BOD contributions to treatment wetland surface water, which varied substantially between and along monitored transects. Because so few studies have investigated treatment dynamics within treatment wetland cells, focusing solely on influent/effluent characterization, radical spatiotemporal variability may be the norm as opposed to the commonly accepted assumptions of relatively uniform pollutant degradation across treatment wetland cells. This spatiotemporal variability in WQPs underscores the dynamic nature of treatment wetlands and the need for routine maintenance, including sludge removal and plant harvesting.

Potential Susceptibility of Six Aquatic Plant Species to Infection by Five Species of Phytophthora.

Investigations of the susceptibility of aquatic plants to species of Phytophthora are limited. Therefore, the objective of this study was to assess the potential susceptibility of six aquatic plant species, frequently used in constructed wetlands or vegetated channels, to infection by five species of Phytophthora commonly found at nurseries in the southeastern United States. In a greenhouse experiment, roots of each plant species (Agrostis alba, Carex stricta, Iris ensata 'Rising Sun', Panicum virgatum, Pontederia cordata, and Typha latifolia) growing in aqueous solutions were exposed to zoospores of each of the species of Phytophthora (Phytophthora cinnamomi, Phytophthora citrophthora, Phytophthora cryptogea, Phytophthora nicotianae, and Phytophthora palmivora). Zoospore presence and activity in solution were monitored with a standard baiting bioassay with rhododendron leaf discs as baits. Experiments were initiated in 2016 and repeated in 2017 and 2018. During the 2016 trials, Phytophthora spp. were not isolated from the roots of any of the plants, but some roots of C. stricta, P. virgatum, and T. latifolia were infected with multiple species of Phytophthora during trials in 2017 and 2018. Presence of plant roots reduced the percentage of rhododendron leaf discs infected by zoospores of four of the species of Phytophthora but not those infected by P. cinnamomi, which suggested that roots of these plants negatively affected the presence or activity of zoospores of these four species of Phytophthora in the aqueous growing solution. Results from this study demonstrated that certain aquatic plant species may be sources of inoculum at ornamental plant nurseries if these plants are present naturally in waterways or used in constructed wetlands treating water flowing off production areas, which could be of concern to plant producers who recycle irrigation water.

Within and between breeding-season changes in contaminant occurrence and body condition in the Antarctic breeding south polar skua.

The Antarctic ecosystem represents a remote region far from point sources of pollution. Still, Antarctic marine predators, such as seabirds, are exposed to organohalogen contaminants (OHCs) which may induce adverse health effects. With increasing restrictions and regulations on OHCs, the levels and exposure are expected to decrease over time. We studied south polar skua (Catharacta maccormiciki), a top predator seabird, to compare OHC concentrations measured in whole blood from 2001/2002 and 2013/2014 in Dronning Maud Land. As a previous study found increasing organochlorine concentrations with sampling day during the 2001/2002 breeding season, suggesting dietary changes, we investigated if this increase was repeated in the 2013/2014 breeding season. In addition to organochlorines, we analyzed hydroxy-metabolites, brominated contaminants and per- and polyfluoroalkyl substances (PFAS) in 2013/2014, as well as dietary descriptors of stable isotopes of carbon and nitrogen, to assess potential changes in diet during breeding. Lipid normalized concentrations of individual OHCs were 63%, 87% and 105% higher for hexachlorobenzene (HCB), 1,1-dichloro-2,2-bis (p-chlorophenyl)ethylene (p,p'-DDE), and ∑Polychlorinated biphenyls (PCBs), respectively, in 2013/2014 compared to 2001/2002. South polar skuas males in 2013/2014 were in poorer body condition than in 2001/2002, and with higher pollutant levels. Poorer body condition may cause the remobilization of contaminants from stored body reserves, and continued exposure to legacy contaminants at overwintering areas may explain the unexpected higher OHC concentrations in 2013/2014 than 2001/2002. Concentrations of protein-associated PFAS increased with sampling day during the 2013/2014 breeding season, whereas the lipid-soluble chlorinated pesticides, PCBs and polybrominated diphenyl ether (PBDEs) showed no change. OHC occurrence was not correlated with stable isotopes. The PFAS biomagnification through the local food web at the colony should be investigated further.

Adaptation of pancreatic cancer cells to nutrient deprivation is reversible and requires glutamine synthetase stabilization by mTORC1.

Pancreatic ductal adenocarcinoma (PDA) is a lethal, therapy-resistant cancer that thrives in a highly desmoplastic, nutrient-deprived microenvironment. Several studies investigated the effects of depriving PDA of either glucose or glutamine alone. However, the consequences on PDA growth and metabolism of limiting both preferred nutrients have remained largely unknown. Here, we report the selection for clonal human PDA cells that survive and adapt to limiting levels of both glucose and glutamine. We find that adapted clones exhibit increased growth in vitro and enhanced tumor-forming capacity in vivo. Mechanistically, adapted clones share common transcriptional and metabolic programs, including amino acid use for de novo glutamine and nucleotide synthesis. They also display enhanced mTORC1 activity that prevents the proteasomal degradation of glutamine synthetase (GS), the rate-limiting enzyme for glutamine synthesis. This phenotype is notably reversible, with PDA cells acquiring alterations in open chromatin upon adaptation. Silencing of GS suppresses the enhanced growth of adapted cells and mitigates tumor growth. These findings identify nongenetic adaptations to nutrient deprivation in PDA and highlight GS as a dependency that could be targeted therapeutically in pancreatic cancer patients.

A Prospective Analysis of Circulating Plasma Metabolites Associated with Ovarian Cancer Risk.

Ovarian cancer has few known risk factors, hampering identification of high-risk women. We assessed the association of prediagnostic plasma metabolites (N = 420) with risk of epithelial ovarian cancer, including both borderline and invasive tumors. A total of 252 cases and 252 matched controls from the Nurses' Health Studies were included. Multivariable logistic regression was used to estimate ORs and 95% confidence intervals (CI), comparing the 90th-10th percentile in metabolite levels, using the permutation-based Westfall and Young approach to account for testing multiple correlated hypotheses. Weighted gene coexpression network analysis (WGCNA; n = 10 metabolite modules) and metabolite set enrichment analysis (n = 23 metabolite classes) were also evaluated. An increase in pseudouridine levels from the 10th to the 90th percentile was associated with a 2.5-fold increased risk of overall ovarian cancer (OR = 2.56; 95% CI, 1.48-4.45; P = 0.001/adjusted P = 0.15); a similar risk estimate was observed for serous/poorly differentiated tumors (n = 176 cases; comparable OR = 2.38; 95% CI, 1.33-4.32; P = 0.004/adjusted P = 0.55). For nonserous tumors (n = 34 cases), pseudouridine and C36:2 phosphatidylcholine plasmalogen had the strongest statistical associations (OR = 9.84; 95% CI, 2.89-37.82; P < 0.001/adjusted P = 0.07; and OR = 0.11; 95% CI, 0.03-0.35; P < 0.001/adjusted P = 0.06, respectively). Five WGCNA modules and 9 classes were associated with risk overall at FDR ≤ 0.20. Triacylglycerols (TAG) showed heterogeneity by tumor aggressiveness (case-only heterogeneity P < 0.0001). The TAG association with risk overall and serous tumors differed by acyl carbon content and saturation. In summary, this study suggests that pseudouridine may be a novel risk factor for ovarian cancer and that TAGs may also be important, particularly for rapidly fatal tumors, with associations differing by structural features. SIGNIFICANCE: Pseudouridine represents a potential novel risk factor for ovarian cancer and triglycerides may be important particularly in rapidly fatal ovarian tumors.

Bacteroides-Derived Sphingolipids Are Critical for Maintaining Intestinal Homeostasis and Symbiosis.

Sphingolipids are structural membrane components and important eukaryotic signaling molecules. Sphingolipids regulate inflammation and immunity and were recently identified as the most differentially abundant metabolite in stool from inflammatory bowel disease (IBD) patients. Commensal bacteria from the Bacteroidetes phylum also produce sphingolipids, but the impact of these metabolites on host pathways is largely uncharacterized. To determine whether bacterial sphingolipids modulate intestinal health, we colonized germ-free mice with a sphingolipid-deficient Bacteroides thetaiotaomicron strain. A lack of Bacteroides-derived sphingolipids resulted in intestinal inflammation and altered host ceramide pools in mice. Using lipidomic analysis, we described a sphingolipid biosynthesis pathway and revealed a variety of Bacteroides-derived sphingolipids including ceramide phosphoinositol and deoxy-sphingolipids. Annotating Bacteroides sphingolipids in an IBD metabolomic dataset revealed lower abundances in IBD and negative correlations with inflammation and host sphingolipid production. These data highlight the role of bacterial sphingolipids in maintaining homeostasis and symbiosis in the gut.

Effects of Maternal Reproductive Investment on Sex-Specific Pollutant Accumulation in Seabirds: A Meta-Analysis.

Avian egg production demands resources such as lipids and proteins. Relative egg size and mass varies across species, reflecting differences in maternal investment. This variability may affect the maternal transfer of anthropogenic pollutants including lipophilic polychlorinated biphenyls (PCBs) and protein-associated per- and polyfluoroalkyl substances (PFASs) and mercury (Hg). We conducted a meta-analysis on seabirds and investigated whether interspecies variation in maternal investment contributes toward skewed pollutant concentration ratios between males and females, as Cmale/Cfemale (80 studies). Overall concentrations of PCBs and perfluorooctanesulfonic acid (PFOS) were 1.6 and 1.3 times higher, respectively, in males than females, whereas mercury was similar between sexes. Few studies compared females and eggs ( n = 6), highlighting a knowledge gap. We found that an increasing maternal investment as a clutch-to-female mass ratio resulted in lower PCB concentrations in females than in males during the incubation period, but no sex-specific differences were observed for mercury and PFOS. Egg production is both a lipid dominated and protein-limited process. Females transfer lipophilic pollutants more easily to eggs, and to a higher degree with increasing maternal investment, but feeding ecology may be more important. Interspecies variation in maternal pollutant transfer may lead to negative effects scaling from an offspring to population level.

Potential Effect of Migration Strategy on Pollutant Occurrence in Eggs of Arctic Breeding Barnacle Geese ( Branta leucopsis).

Arctic-breeding geese acquire resources for egg production from overwintering grounds, spring stopover sites and breeding grounds, where pollutant exposure may differ. We investigated the effect of migration strategy on pollutant occurrence of lipophilic polychlorinated biphenyls (PCBs) and protein-associated poly- and perfluoroalkyl substances (PFASs) and mercury (Hg) in eggs of herbivorous barnacle geese ( Branta leucopsis) from an island colony on Svalbard. Stable isotopes (δ13C and δ15N) in eggs and vegetation collected along the migration route were similar. Pollutant concentrations in eggs were low, reflecting their terrestrial diet (∑PCB = 1.23 ± 0.80 ng/g ww; ∑PFAS = 1.21 ± 2.97 ng/g ww; Hg = 20.17 ± 7.52 ng/g dw). PCB concentrations in eggs increased with later hatch date, independent of lipid content which also increased over time. Some females may remobilize and transfer more PCBs to their eggs, by delaying migration several weeks, relying on more polluted and stored resources, or being in poor body condition when arriving at the breeding grounds. PFAS and Hg occurrence in eggs did not change throughout the breeding season, suggesting migration has a greater effect on lipophilic pollutants. Pollutant exposure during offspring production in arctic-breeding migrants may result in different profiles, with effects becoming more apparent with increasing trophic levels.

Ablation of insulin receptor substrates 1 and 2 suppresses Kras-driven lung tumorigenesis.

Non-small-cell lung cancer (NSCLC) is a leading cause of cancer death worldwide, with 25% of cases harboring oncogenic Kirsten rat sarcoma (KRAS). Although KRAS direct binding to and activation of PI3K is required for KRAS-driven lung tumorigenesis, the contribution of insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) in the context of mutant KRAS remains controversial. Here, we provide genetic evidence that lung-specific dual ablation of insulin receptor substrates 1/2 (Irs1/Irs2), which mediate insulin and IGF1 signaling, strongly suppresses tumor initiation and dramatically extends the survival of a mouse model of lung cancer with Kras activation and p53 loss. Mice with Irs1/Irs2 loss eventually succumb to tumor burden, with tumor cells displaying suppressed Akt activation and strikingly diminished intracellular levels of essential amino acids. Acute loss of IRS1/IRS2 or inhibition of IR/IGF1R in KRAS-mutant human NSCLC cells decreases the uptake and lowers the intracellular levels of amino acids, while enhancing basal autophagy and sensitivity to autophagy and proteasome inhibitors. These findings demonstrate that insulin/IGF1 signaling is required for KRAS-mutant lung cancer initiation, and identify decreased amino acid levels as a metabolic vulnerability in tumor cells with IR/IGF1R inhibition. Consequently, combinatorial targeting of IR/IGF1R with autophagy or proteasome inhibitors may represent an effective therapeutic strategy in KRAS-mutant NSCLC.

Critical role for arginase 2 in obesity-associated pancreatic cancer.

Obesity is an established risk factor for pancreatic ductal adenocarcinoma (PDA). Despite recent identification of metabolic alterations in this lethal malignancy, the metabolic dependencies of obesity-associated PDA remain unknown. Here we show that obesity-driven PDA exhibits accelerated growth and a striking transcriptional enrichment for pathways regulating nitrogen metabolism. We find that the mitochondrial form of arginase (ARG2), which hydrolyzes arginine into ornithine and urea, is induced upon obesity, and silencing or loss of ARG2 markedly suppresses PDA. In vivo infusion of 15N-glutamine in obese mouse models of PDA demonstrates enhanced nitrogen flux into the urea cycle and infusion of 15N-arginine shows that Arg2 loss causes significant ammonia accumulation that results from the shunting of arginine catabolism into alternative nitrogen repositories. Furthermore, analysis of PDA patient tumors indicates that ARG2 levels correlate with body mass index (BMI). The specific dependency of PDA on ARG2 rather than the principal hepatic enzyme ARG1 opens a therapeutic window for obesity-associated pancreatic cancer.Obesity is an established risk factor for pancreatic ductal adenocarcinoma (PDA). Here the authors show that obesity induces the expression of the mitochondrial form of arginase ARG2 in PDA and that ARG2 silencing or loss results in ammonia accumulation and suppression of obesity-driven PDA tumor growth.

Water Use and Treatment in Container-Grown Specialty Crop Production: A Review.

While governments and individuals strive to maintain the availability of high-quality water resources, many factors can "change the landscape" of water availability and quality, including drought, climate change, saltwater intrusion, aquifer depletion, population increases, and policy changes. Specialty crop producers, including nursery and greenhouse container operations, rely heavily on available high-quality water from surface and groundwater sources for crop production. Ideally, these growers should focus on increasing water application efficiency through proper construction and maintenance of irrigation systems, and timing of irrigation to minimize water and sediment runoff, which serve as the transport mechanism for agrichemical inputs and pathogens. Rainfall and irrigation runoff from specialty crop operations can contribute to impairment of groundwater and surface water resources both on-farm and into the surrounding environment. This review focuses on multiple facets of water use, reuse, and runoff in nursery and greenhouse production including current and future regulations, typical water contaminants in production runoff and available remediation technologies, and minimizing water loss and runoff (both on-site and off-site). Water filtration and treatment for the removal of sediment, pathogens, and agrichemicals are discussed, highlighting not only existing understanding but also knowledge gaps. Container-grown crop producers can either adopt research-based best management practices proactively to minimize the economic and environmental risk of limited access to high-quality water, be required to change by external factors such as regulations and fines, or adapt production practices over time as a result of changing climate conditions.

Starved epithelial cells uptake extracellular matrix for survival.

Extracellular matrix adhesion is required for normal epithelial cell survival, nutrient uptake and metabolism. This requirement can be overcome by oncogene activation. Interestingly, inhibition of PI3K/mTOR leads to apoptosis of matrix-detached, but not matrix-attached cancer cells, suggesting that matrix-attached cells use alternate mechanisms to maintain nutrient supplies. Here we demonstrate that under conditions of dietary restriction or growth factor starvation, where PI3K/mTOR signalling is decreased, matrix-attached human mammary epithelial cells upregulate and internalize ß4-integrin along with its matrix substrate, laminin. Endocytosed laminin localizes to lysosomes, results in increased intracellular levels of essential amino acids and enhanced mTORC1 signalling, preventing cell death. Moreover, we show that starved human fibroblasts secrete matrix proteins that maintain the growth of starved mammary epithelial cells contingent upon epithelial cell ß4-integrin expression. Our study identifies a crosstalk between stromal fibroblasts and epithelial cells under starvation that could be exploited therapeutically to target tumours resistant to PI3K/mTOR inhibition.

Single and mixture effects of aquatic micropollutants studied in precision-cut liver slices of Atlantic cod (Gadus morhua).

The low concentrations of most contaminants in the aquatic environment individually may not affect the normal function of the organisms on their own. However, when combined, complex mixtures may provoke unexpected effects even at low amounts. Selected aquatic micropollutants such as chlorpyrifos, bis-(2-ethylhexyl)-phthalate (DEHP), perfluorooctanoic acid (PFOA) and 17α-ethinylestradiol (EE2) were tested singly and in mixtures at nM to µM concentrations using precision-cut liver slices (PCLS) of Atlantic cod (Gadus morhua). Fish liver is a target organ for contaminants due to its crucial role in detoxification processes. In order to understand the effects on distinct key liver metabolic pathways, transcription levels of various genes were measured, including cyp1a1 and cyp3a, involved in the metabolism of organic compounds, including toxic ones, and the catabolism of bile acids and steroid hormones; cyp7a1, fabp and hmg-CoA, involved in lipid and cholesterol homeostasis; cyp24a1, involved in vitamin D metabolism; and vtg, a key gene in xenoestrogenic response. Only EE2 had significant effects on gene expression in cod liver slices when exposed singly at the concentrations tested. However, when exposed in combinations, effects not detected in single exposure conditions arose, suggesting complex interactions between studied pollutants that could not be predicted from the results of individual exposure scenarios. Thus, the present work highlights the importance of assessing mixtures when describing the toxic effects of micropollutants to fish liver metabolism.

Structure of N-formimino-L-glutamate iminohydrolase from Pseudomonas aeruginosa.

N-Formimino-l-glutamate iminohydrolase (HutF), from Pseudomonas aeruginosa with a locus tag of Pa5106 ( gi|15600299 ), is a member of the amidohydrolase superfamily. This enzyme catalyzes the deamination of N-formimino-l-glutamate to N-formyl-l-glutamate and ammonia in the histidine degradation pathway. The crystal structure of Pa5106 was determined in the presence of the inhibitors N-formimino-l-aspartate and N-guanidino-l-glutaric acid at resolutions of 1.9 and 1.4 Å, respectively. The structure of an individual subunit is composed of two domains with the larger domain folding as a distorted (ß/α)8-barrel. The (ß/α)8-barrel domain is composed of eight ß-strands flanked by 11 α-helices, whereas the smaller domain is made up of eight ß-strands. The active site of Pa5106 contains a single zinc atom that is coordinated by His-56, His-58, His-232, and Asp-320. The nucleophilic solvent water molecule coordinates with the zinc atom at a distance of 2.0 Å and is hydrogen bonded to Asp-320 and His-269. The α-carboxylate groups of both inhibitors are hydrogen bonded to the imidazole moiety of His-206, the hydroxyl group of Tyr-121, and the side chain amide group of Gln-61. The side chain carboxylate groups of the two inhibitors are ion-paired with the guanidino groups of Arg-209 and Arg-82. Computational docking of high-energy tetrahedral intermediate forms of the substrate, N-formimino-l-glutamate, to the three-dimensional structure of Pa5106 suggests that this compound likely undergoes a re-faced nucleophilic attack at the formimino group by the metal-bound hydroxide. A catalytic mechanism of the reaction catalyzed by Pa5106 is proposed.

Discovery of a bacterial 5-methylcytosine deaminase.

5-Methylcytosine is found in all domains of life, but the bacterial cytosine deaminase from Escherichia coli (CodA) will not accept 5-methylcytosine as a substrate. Since significant amounts of 5-methylcytosine are produced in both prokaryotes and eukaryotes, this compound must eventually be catabolized and the fragments recycled by enzymes that have yet to be identified. We therefore initiated a comprehensive phylogenetic screen for enzymes that may be capable of deaminating 5-methylcytosine to thymine. From a systematic analysis of sequence homologues of CodA from thousands of bacterial species, we identified putative cytosine deaminases where a "discriminating" residue in the active site, corresponding to Asp-314 in CodA from E. coli, was no longer conserved. Representative examples from Klebsiella pneumoniae (locus tag: Kpn00632), Rhodobacter sphaeroides (locus tag: Rsp0341), and Corynebacterium glutamicum (locus tag: NCgl0075) were demonstrated to efficiently deaminate 5-methylcytosine to thymine with values of kcat/Km of 1.4 × 10(5), 2.9 × 10(4), and 1.1 × 10(3) M(-1) s(-1), respectively. These three enzymes also catalyze the deamination of 5-fluorocytosine to 5-fluorouracil with values of kcat/Km of 1.2 × 10(5), 6.8 × 10(4), and 2.0 × 10(2) M(-1) s(-1), respectively. The three-dimensional structure of Kpn00632 was determined by X-ray diffraction methods with 5-methylcytosine (PDB id: 4R85 ), 5-fluorocytosine (PDB id: 4R88 ), and phosphonocytosine (PDB id: 4R7W ) bound in the active site. When thymine auxotrophs of E. coli express these enzymes, they are capable of growth in media lacking thymine when supplemented with 5-methylcytosine. Expression of these enzymes in E. coli is toxic in the presence of 5-fluorocytosine, due to the efficient transformation to 5-fluorouracil.

Substrate deconstruction and the nonadditivity of enzyme recognition.

Predicting substrates for enzymes of unknown function is a major postgenomic challenge. Substrate discovery, like inhibitor discovery, is constrained by our ability to explore chemotypes; it would be expanded by orders of magnitude if reactive sites could be probed with fragments rather than fully elaborated substrates, as is done for inhibitor discovery. To explore the feasibility of this approach, substrates of six enzymes from three different superfamilies were deconstructed into 41 overlapping fragments that were tested for activity or binding. Surprisingly, even those fragments containing the key reactive group had little activity, and most fragments did not bind measurably, until they captured most of the substrate features. Removing a single atom from a recognized substrate could often reduce catalytic recognition by 6 log-orders. To explore recognition at atomic resolution, the structures of three fragment complexes of the ß-lactamase substrate cephalothin were determined by X-ray crystallography. Substrate discovery may be difficult to reduce to the fragment level, with implications for function discovery and for the tolerance of enzymes to metabolite promiscuity. Pragmatically, this study supports the development of libraries of fully elaborated metabolites as probes for enzyme function, which currently do not exist.

Structure-guided discovery of new deaminase enzymes.

A substantial challenge for genomic enzymology is the reliable annotation for proteins of unknown function. Described here is an interrogation of uncharacterized enzymes from the amidohydrolase superfamily using a structure-guided approach that integrates bioinformatics, computational biology, and molecular enzymology. Previously, Tm0936 from Thermotoga maritima was shown to catalyze the deamination of S-adenosylhomocysteine (SAH) to S-inosylhomocysteine (SIH). Homologues of Tm0936 homologues were identified, and substrate profiles were proposed by docking metabolites to modeled enzyme structures. These enzymes were predicted to deaminate analogues of adenosine including SAH, 5'-methylthioadenosine (MTA), adenosine (Ado), and 5'-deoxyadenosine (5'-dAdo). Fifteen of these proteins were purified to homogeneity, and the three-dimensional structures of three proteins were determined by X-ray diffraction methods. Enzyme assays supported the structure-based predictions and identified subgroups of enzymes with the capacity to deaminate various combinations of the adenosine analogues, including the first enzyme (Dvu1825) capable of deaminating 5'-dAdo. One subgroup of proteins, exemplified by Moth1224 from Moorella thermoacetica, deaminates guanine to xanthine, and another subgroup, exemplified by Avi5431 from Agrobacterium vitis S4, deaminates two oxidatively damaged forms of adenine: 2-oxoadenine and 8-oxoadenine. The sequence and structural basis of the observed substrate specificities were proposed, and the substrate profiles for 834 protein sequences were provisionally annotated. The results highlight the power of a multidisciplinary approach for annotating enzymes of unknown function.