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

Abstract

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.

Crystal structure ofEscherichia colipurine nucleoside phosphorylase complexed with acyclovir

“…4b). In human PNP, Asn243 is analogous to the essential active-site residue Asp204 found in E. coli PNP that is essential for the activity and specificity of the enzyme (dos Santos et al, 2003;Mahor et al, 2016). The O6 and N7 atoms of acyclovir form direct bonds to OD1 and ND2 of Asn243 in the human enzyme, whereas in the E. coli enzyme the bonds between these atoms and Asp204 are absent (at least in two subunits).…”

A transcript-wide association study in physical activity intervention implicates molecular pathways in chronic disease

“…High expression levels of PNP -a gene responsible for producing purine nucleoside phosphorylase enzyme -have been associated with higher purine content in the body 86 , a condition associated with increased risk of hyperuricemia and gout. Hyperuricemia and gout are known risk factors for cardiovascular disease, renal disease, insulin resistance, obesity and other metabolic diseases [87][88][89][90] . Given that decreased PNP is associated with decreased BMI in our twins cohort, we hypothesize that physical activity may play a role in the reduction of adiposity through regulation of the purine metabolism pathway.…”

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

“…The low purine diet recommendation is promising and safe, but it eliminates a wide range of foods and beverages, which makes therapy difficult to be adopted by a major percentage of patients. One of the strategies to make this therapy safe and attractive is to produce the foods and beverages that are low in purine content through the enzymatic application (Trautwein-Schult et al, 2014 ; Jankowska et al, 2015 ; Mahor et al, 2016 ). This gives the patients an option to choose a wide variety of low purine content foods and beverages without any risk of hyperuricemia and gout.…”

“…Our group is working on the enzymes involved in the purine degradation pathway of K. lactis , an industrially important and Food and Drug Administration (FDA)-approved generally regarded as safe (GRAS) status yeast. We have earlier worked on uricase and purine nucleoside phosphorylase of this organism (Kumar, 2012 ; Mahor et al, 2016 ). In the present study, we mainly focused on adenine deaminase and guanine deaminase enzymes of the purine degradation pathway of K. lactis and their application in lowering purine content in beverages.…”

“…Previously, we have overexpressed and purified purine nucleoside phosphorylase (Mahor et al, 2016 ) and xanthine dioxygenase (Mahor et al, 2016 , unpublished data) enzymes from K. lactis . Using all these enzymes, we demonstrated that applying an enzyme cocktail ( Klac ADA, Klac GDA, Klac PNP, and Klac XanA) is able to reduce purines viz.…”