creators_name: Kunze, Gotthard
creators_name: Mahor, Durga
creators_name: Priyanka, Anu
creators_name: Prasad, Gandham S
creators_name: Thakur, Krishan Gopal
type: article
datestamp: 2018-03-24 04:16:54
lastmod: 2018-03-24 04:16:54
metadata_visibility: show
title: Functional and Structural Characterization of Purine Nucleoside Phosphorylase from Kluyveromyces lactis and Its Potential Applications in Reducing Purine Content in Food
ispublished: pub
subjects: QR
keywords: Amino Acid Sequence
Catalytic Domain
Cloning, Molecular
Crystallography, X-Ray
Enzyme Stability
note: Open Access
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.
date: 2016
date_type: published
publication: PLOS ONE
volume: 11
number: 10
publisher: Public Library of Science
pagerange: e0164279
id_number: doi:10.1371/journal.pone.0164279
refereed: TRUE
issn: 1932-6203
official_url: http://dx.doi.org/10.1371/journal.pone.0164279
citation:   Kunze, Gotthard and Mahor, Durga and Priyanka, Anu and Prasad, Gandham S and Thakur, Krishan Gopal  (2016) Functional and Structural Characterization of Purine Nucleoside Phosphorylase from Kluyveromyces lactis and Its Potential Applications in Reducing Purine Content in Food.  PLOS ONE, 11 (10).  e0164279.  ISSN 1932-6203