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

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

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

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

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

Valorization of Small Alkanes by Biocatalytic Oxyfunctionalization

The oxidation of alkanes into valuable chemical products is a vital reaction in organic synthesis. This reaction, however, is challenging, owing to the inertness of C−H bonds. Transition metal catalysts for C−H functionalization are frequently explored. Despite chemical alternatives, nature has also evolved powerful oxidative enzymes (e. g., methane monooxygenases, cytochrome P450 oxygenases, peroxygenases) that are capable of transforming C−H bonds under very mild conditions, with only the use of molecular oxygen or hydrogen peroxide as electron acceptors. Although progress in alkane oxidation has been reviewed extensively, little attention has been paid to small alkane oxidation. The latter holds great potential for the manufacture of chemicals. This Minireview provides a concise overview of the most relevant enzyme classes capable of small alkanes (C&lt;6) oxyfunctionalization, describes the essentials of the catalytic mechanisms, and critically outlines the current state-of-the-art in preparative applications.Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.BT/Biocatalysi

Active site architecture of <i>Klac</i>PNP with bound hypoxanthine in the active site.

<p>(A) ESI-MS analysis of intrinsically bound ligand (upper panel) and expected mass of the possible ligands (lower panel) is shown. Mass spectrometry analysis unambiguously suggests that hypoxanthine is bound to the active site. (B) Possible conventional hydrogen bond and weak C–H⋯O interactions are represented by broken black and broken red lines, respectively. The Phe213 side chain is oriented almost perpendicular to the substrate and mediates the π-π interactions. Hypoxanthine, amino acids, and water molecules are represented as ball and stick, sticks, and red spheres, respectively. The atoms are colored according to the following color code: carbon, grey; nitrogen, blue; oxygen, red.</p

HPLC analysis of the beer sample before and after the enzyme treatment.

<p>(A) The chromatogram shows purine content in the beer sample before (dotted back line) and after <i>Klac</i>PNP enzymatic treatment (solid grey line). (B) The chromatogram shows purine content in the beer sample with added adenosine (20 mg/L) before (dotted back line) and after <i>Klac</i>PNP^N256D mutant enzyme reaction (solid grey line). Beer sample treated with 0.8 U of <i>Klac</i>PNP and <i>Klac</i>PNP^N256D mutant. In the panel (A) there is a decrease in the intensity of the inosine peak (4) and an increase in the hypoxanthine peak intensity (3). In the panel (B), there was a decrease in the inosine (4) and adenosine (5) peak intensities and corresponding increase in the hypoxanthine (3) and adenine (1) peak intensities, suggesting the conversion of inosine and adenosine into hypoxanthine and adenine, respectively. (C) ESI-MS analysis performed for the peak fractions. Expected and observed molecular mass of adenine, guanine, hypoxanthine, inosine, and adenosine are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164279#pone.0164279.t003" target="_blank">Table 3</a>.</p

Structural similarity between <i>Klac</i>PNP with human and <i>E</i>. <i>coli</i> PNP.

<p>(A and B) Structural superimpositions in putty representation showing the structural homology of <i>Klac</i>PNP monomer with human PNP and <i>E</i>. <i>coli</i> PNP. The thick regions in the putty, localized primarily at high B-factor loop regions, have higher structural deviations. The large differences in the loop regions and presence of an extra helix at the N-terminal region of <i>Klac</i>PNP can be observed in the panel.</p

Purine content in the beer sample before and after enzymatic treatments.

<p>Purine content in the beer sample before and after enzymatic treatments.</p