Biochemical Characterization of Kluyveromyces lactis Adenine Deaminase and Guanine Deaminase and Their Potential Application in Lowering Purine Content in Beer

Excess amounts of uric acid in humans leads to hyperuricemia, which is a biochemical precursor of gout and is also associated with various other disorders. Gout is termed as crystallization of uric acid, predominantly within joints. The burden of hyperuricemia and gout has increased worldwide due to lifestyle changes, obesity, and consumption of purine-rich foods, fructose-containing drinks, and alcoholic beverages. Some of the therapies available to cure gout are associated with unwanted side-effects and antigenicity. We propose an attractive and safe strategy to reduce purine content in beverages using enzymatic application of purine degrading enzymes such as adenine deaminase (ADA) and guanine deaminase (GDA) that convert adenine and guanine into hypoxanthine and xanthine, respectively. We cloned, expressed, purified, and biochemically characterized both adenine deaminase (ADA) and guanine deaminase (GDA) enzymes that play important roles in the purine degradation pathway of Kluyveromyces lactis, and demonstrate their application in lowering purine content in a beverage. The popular beverage beer has been selected as an experimental sample as it confers higher risks of hyperuricemia and gout. Quantification of purine content in 16 different beers from the Indian market showed varying concentrations of different purines. Enzymatic treatment of beer samples with ADA and GDA showed a reduction of adenine and guanine content, respectively. These enzymes in combination with other purine degrading enzymes showed marked reduction in purine content in beer samples. Both enzymes can work at 5.0–8.0 pH range and retain >50% activity at 40°C, making them good candidates for industrial applications

Uric acid in men with acute stroke

Abstract Higher levels of uric acid in men as compared to women can be a reason behind greater incidence of stroke in men. The objective of the present study was to evaluate the levels of uric acid in men with acute stroke and correlate with stroke severity.For the purpose of the study ,50 male patients of acute stroke admitted to the hospital and 50 age matched healthy controls were included in the study. Routine biochemical parameters including fasting blood glucose, uric acid and lipid profile were assessed in serum obtained from 5 ml of fasting blood sample. Patients with kidney or liver diseases, malignancies, diuretic use, alcohol intake, on iron or antioxidant therapy were excluded from the study. Initial stroke severity was measured by the National Institute of Health Stroke (NIHS) scale. It was found that , among the 50 cases, 38(76%) had ischemic stroke and 12(24%) had hemorrhagic stroke. Serum uric acid levels were very significantly higher in cases (p<0.001) than controls. There was strong positive correlation between uric acid levels and initial stroke severity (p=0.006, r=0.386). Also, serum uric acid showed a statistically significant correlation with fasting blood glucose, TG and VLDL and an inverse association with HDL in both cases and controls. The conclusion drawn was that the significantly higher levels of uric acid in men with stroke and the positive association of uric acid with stroke severity suggest a possible role of uric acid as a risk factor for stroke in men

FACTIFY3M: A Benchmark for Multimodal Fact Verification with Explainability through 5W Question-Answering

Combating disinformation is one of the burning societal crises -- about 67% of the American population believes that disinformation produces a lot of uncertainty, and 10% of them knowingly propagate disinformation. Evidence shows that disinformation can manipulate democratic processes and public opinion, causing disruption in the share market, panic and anxiety in society, and even death during crises. Therefore, disinformation should be identified promptly and, if possible, mitigated. With approximately 3.2 billion images and 720,000 hours of video shared online daily on social media platforms, scalable detection of multimodal disinformation requires efficient fact verification. Despite progress in automatic text-based fact verification (e.g., FEVER, LIAR), the research community lacks substantial effort in multimodal fact verification. To address this gap, we introduce FACTIFY 3M, a dataset of 3 million samples that pushes the boundaries of the domain of fact verification via a multimodal fake news dataset, in addition to offering explainability through the concept of 5W question-answering. Salient features of the dataset include: (i) textual claims, (ii) ChatGPT-generated paraphrased claims, (iii) associated images, (iv) stable diffusion-generated additional images (i.e., visual paraphrases), (v) pixel-level image heatmap to foster image-text explainability of the claim, (vi) 5W QA pairs, and (vii) adversarial fake news stories.Comment: arXiv admin note: text overlap with arXiv:2305.0432

Functional and Structural Characterization of Purine Nucleoside Phosphorylase from Kluyveromyces lactis and Its Potential Applications in Reducing Purine Content in Food.

Consumption of foods and beverages with high purine content increases the risk of hyperuricemia, which causes gout and can lead to cardiovascular, renal, and other metabolic disorders. As patients often find dietary restrictions challenging, enzymatically lowering purine content in popular foods and beverages offers a safe and attractive strategy to control hyperuricemia. Here, we report structurally and functionally characterized purine nucleoside phosphorylase (PNP) from Kluyveromyces lactis (KlacPNP), a key enzyme involved in the purine degradation pathway. We report a 1.97 Å resolution crystal structure of homotrimeric KlacPNP with an intrinsically bound hypoxanthine in the active site. KlacPNP belongs to the nucleoside phosphorylase-I (NP-I) family, and it specifically utilizes 6-oxopurine substrates in the following order: inosine > guanosine > xanthosine, but is inactive towards adenosine. To engineer enzymes with broad substrate specificity, we created two point variants, KlacPNPN256D and KlacPNPN256E, by replacing the catalytically active Asn256 with Asp and Glu, respectively, based on structural and comparative sequence analysis. KlacPNPN256D not only displayed broad substrate specificity by utilizing both 6-oxopurines and 6-aminopurines in the order adenosine > inosine > xanthosine > guanosine, but also displayed reversal of substrate specificity. In contrast, KlacPNPN256E was highly specific to inosine and could not utilize other tested substrates. Beer consumption is associated with increased risk of developing gout, owing to its high purine content. Here, we demonstrate that KlacPNP and KlacPNPN256D could be used to catalyze a key reaction involved in lowering beer purine content. Biochemical properties of these enzymes such as activity across a wide pH range, optimum activity at about 25°C, and stability for months at about 8°C, make them suitable candidates for food and beverage industries. Since KlacPNPN256D has broad substrate specificity, a combination of engineered KlacPNP and other enzymes involved in purine degradation could effectively lower the purine content in foods and beverages

HPLC chromatogram of different substrates in the presence of <i>Klac</i>PNP.

<p>(A) Chromatogram showing the retention profile of inosine (solid black line) and hypoxanthine (dotted grey line) standards, and inosine + <i>Klac</i>PNP (solid grey line). (B) Chromatogram showing the retention profile for guanosine (solid grey line), guanine (dotted grey line), and guanosine + <i>Klac</i>PNP (solid black line). (C) Xanthosine (solid black line), xanthine (dotted grey line), and xanthosine + <i>Klac</i>PNP (solid grey line). (D) Adenosine (solid black line), adenine (dotted grey line), and adenosine + <i>Klac</i>PNP (solid black line). Adenosine with <i>Klac</i>PNP enzyme reaction showed no consumption of adenosine.</p

Multiple sequence alignment of <i>Klac</i>PNP homologs.

<p>Sequence comparison of <i>Klac</i>PNP with homotrimeric PNPs (specific for 6-oxopurines: <i>S</i>. <i>cerevisiae</i>, human, and calf spleen PNPs), homohexameric PNPs (specific for 6-aminopurines: <i>B</i>. <i>cereus</i>, <i>B</i>. <i>subtilis</i>, and <i>E</i>. <i>coli</i> PNPs). Highly conserved regions are highlighted with red boxes; conservative substitutions are also boxed. The figure was drawn by using the ESPript 3 server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164279#pone.0164279.ref059" target="_blank">59</a>]. Active site residues involved in the interaction with hypoxanthine are shown with an asterisk and the catalytically active residue that is known to play an important role in substrate specificity is shown with a red filled circle [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164279#pone.0164279.ref045" target="_blank">45</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164279#pone.0164279.ref049" target="_blank">49</a>].</p

Comparative structural analysis of the active sites of PNPs.

<p>Crystal structures of <i>K</i>. <i>lactis</i> (green, hypoxanthine bound structure), calf spleen (magenta, PDB ID 1VFN, hypoxanthine bound structure) and human (cyan, PDB ID 1RCT, inosine bound structure) PNPs were used for the structural comparison [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164279#pone.0164279.ref064" target="_blank">64</a>]. The active site is located in the vicinity of the intersubunit interface, and a structurally equivalent Phe172 is contributed from the neighboring monomer. Water molecules at probable phosphate and ribose binding sites in the <i>Klac</i>PNP and the calf spleen PNP are shown as blue and orange spheres, respectively. The two conserved water molecules involved in the water-mediated interactions are encircled (red broken circle). Ligands and amino acids in the active site are shown in the stick representation. Oxygen, nitrogen, and sulfur atoms are shown in red, blue, and yellow colors, respectively. A sulfate ion occupies the potential phosphate binding site. Most of the residues forming the active site superpose well, except for variations in the turn connecting β1 and 3₁₀ helix. For clarity, some residues, which are not a part of the active site, have been removed.</p