Biochemical Characterization of Human Gluconokinase and the Proposed Metabolic Impact of Gluconic Acid as Determined by Constraint Based Metabolic Network Analysis Neha Rohatgi 1,2 , Tine Kragh Nielsen 3 , Sara Petersen Bjørn 3 , Ivar Axelsson 1 , Giuseppe Paglia 1 , Bjørn Gunnar Voldborg 3 , Bernhard O. Palsson 1 ,O ´ ttar Rolfsson 1,2 * 1 Center for Systems Biology, University of Iceland, Reykjavik, Iceland, 2 University of Iceland Biomedical Center, Reykjavik, Iceland, 3 Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark Abstract The metabolism of gluconate is well characterized in prokaryotes where it is known to be degraded following phosphorylation by gluconokinase. Less is known of gluconate metabolism in humans. Human gluconokinase activity was recently identified proposing questions about the metabolic role of gluconate in humans. Here we report the recombinant expression, purification and biochemical characterization of isoform I of human gluconokinase alongside substrate specificity and kinetic assays of the enzyme catalyzed reaction. The enzyme, shown to be a dimer, had ATP dependent phosphorylation activity and strict specificity towards gluconate out of 122 substrates tested. In order to evaluate the metabolic impact of gluconate in humans we modeled gluconate metabolism using steady state metabolic network analysis. The results indicate that significant metabolic flux changes in anabolic pathways linked to the hexose monophosphate shunt (HMS) are induced through a small increase in gluconate concentration. We argue that the enzyme takes part in a context specific carbon flux route into the HMS that, in humans, remains incompletely explored. Apart from the biochemical description of human gluconokinase, the results highlight that little is known of the mechanism of gluconate metabolism in humans despite its widespread use in medicine and consumer products. Citation: Rohatgi N, Nielsen TK, Bjørn SP, Axelsson I, Paglia G, et al. (2014) Biochemical Characterization of Human Gluconokinase and the Proposed Metabolic Impact of Gluconic Acid as Determined by Constraint Based Metabolic Network Analysis. PLoS ONE 9(6): e98760. doi:10.1371/journal.pone.0098760 Editor: Mark R. Muldoon, Manchester University, United Kingdom Received December 19, 2013; Accepted May 6, 2014; Published June 4, 2014 Copyright: ß 2014 Rohatgi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the European Research Council grant proposal no. 232816. The funders had no role in study design, data collection and analysis. Competing Interests: The authors have declared that no competing interests exist. * E-mail: ottarr@hi.is Introduction Gluconate is a C-1 oxidized derivative of glucose, widely distributed in nature and commonly used as an acidity regulator in both food and drugs [1]. Gluconate is an excellent chelator of calcium ions and calcium gluconate is often given intravenously in order to regulate intravenous Ca 2+ levels. While this clinical measure undoubtedly focuses on replenishing Ca 2+ , gluconate and its chemical counterpart gluconolactone against which it exists in chemical equilibrium, have in fact been shown to exhibit antioxidant properties and result in increased plasma levels of glutathione [2]. Lowered plasma levels of gluconate have also been associated with Alzheimer’s disease [3] and increased oxidative stress [4]. We recently highlighted that gluconate metabolism in humans is unaccounted for using a computational network gap filling approach of the human metabolic network Recon 1. Gluconate catabolism was computed to take place through phosphorylation of gluconate to generate 6-phosphogluconate which could then be further degraded through the hexose monophosphate shunt (HMS) via 6-phosphogluconate dehydrogenase [5]. This catabolic route has indeed been shown to take place in rat liver perfusions [6] and corresponds to well researched degradation routes of gluconate in microorganisms. These involve metabolism via (I) direct internalization from the environment, (II) conversion from L-idonic acid or (III) by direct oxidation of glucose via glucono- 1,5-lactone [7–9]. A key enzyme in all the gluconate degradation routes is gluconokinase (GntK) which phosphorylates gluconate at the C-6 position thereby priming its catabolism through the HMS or the Entner-Doudoroff pathway in prokaryotes. The human gene C9orf103 was identified through a metabolic network gap filling effort of Recon 1 and through amino acid sequence alignment as a likely kinase responsible for the initial step in gluconate catabolism in humans [5]. C9orf103 had previously been cloned and sequenced in relation to it being a plausible tumor suppressor gene associated with acute myeloid leukemia [10]. In vitro assays of isoforms I and II of C9orf103 expressed in human HeLa cell lysates showed that only isoform I had ATP dependant phosphorylation activity consistent with the absence of a phosphate binding loop domain in isoform II. Isoform I shows 35% sequence similarity to both GntKs encoded within the E.coli genome. A defining structural difference is an 18 amino acid insert that is found in various NMP kinases that have similar protein PLOS ONE | www.plosone.org 1 June 2014 | Volume 9 | Issue 6 | e98760