1 Unexpected roles for ADH1 and SORD in catalyzing the final step of erythritol biosynthesis Lisa Schlicker 1 , Doletha M. E. Szebenyi 2 , Semira R. Ortiz 3 , Alexander Heinz 1 , Karsten Hiller 1,4 and Martha S. Field 3* From the 1 Department of Bioinformatics and Biochemistry, BRICS, Technische Universität Braunschweig, 38106 Braunschweig, Germany; 2 MacCHESS, Cornell University, Ithaca, NY 14853, USA; 3 Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853, USA; 4 Helmholtz Zentrum für Infektionsforschung, 38124 Braunschweig, Germany Running title: ADH1 and SORD catalyze erythritol synthesis * To whom correspondence should be addressed: Martha S. Field, Division of Nutritional Sciences, Ithaca, NY 14853; mas246@cornell.edu; Tel.(607) 255-6081; Fax. (607) 255-1033 Keywords: erythritol, glucose metabolism, alcohol dehydrogenase 1 (ADH1), sorbitol dehydrogenase (SORD), enzyme kinetics, enzyme catalysis, biomarker ABSTRACT The low-calorie sweetener erythritol is endogenously produced from glucose through the pentose phosphate pathway in humans. Erythritol is of medical interest because elevated plasma levels of this polyol are predictive for visceral adiposity gain and development of type 2 diabetes. However, the mechanisms behind these associations remain unknown because the erythritol biosynthesis pathway, particularly the enzyme catalyzing the final step of erythritol synthesis (reduction of erythrose to erythritol), is not characterized. In this study, we purified two enzymes from rabbit liver capable of catalyzing the conversion of erythrose to erythritol: alcohol dehydrogenase 1 (ADH1) and sorbitol dehydrogenase (SORD). Both recombinant human ADH1 and SORD reduce erythrose to erythritol, using NADPH as a co-factor, and cell culture studies indicate that this activity is primarily NADPH-dependent. We found that ADH1 variants vary markedly in both their affinity for erythrose and their catalytic capacity (turnover number). Interestingly, the recombinant protein produced from the ADH1B2 variant, common in Asian populations, is not active when NADPH is used as a co-factor in vitro. We also confirmed SORD contributes to intracellular erythritol production in human A549 lung cancer cells, where ADH1 is minimally expressed. In summary, human ADH1 and SORD catalyze the conversion of erythrose to erythritol, pointing to novel roles for two dehydrogenase proteins in human glucose metabolism that may contribute to individual responses to diet. Proteomics data are available via ProteomeXchange with identifier PXD015178. ______________________________________ Erythritol is a four carbon polyol which was approved as low calorie sweetener by the FDA in 2001 [1]. It naturally occurs in various alcoholic beverages and fruits and has 70% of the sweetness of sucrose [2]. In a large cohort of college freshmen, Hootman et al. identified elevated plasma erythritol levels as a predictive marker for central adiposity gain and high glycemia [3]. Plasma erythritol was also recently associated with onset of type 2 diabetes mellitus (T2DM) in adults followed over 20 years in the Atherosclerosis Risk in Communities (ARIC) cohort [4]. Erythritol synthesis was not believed to occur in humans [2, 5]. However, using stable isotope tracing, evidence was provided for the first time that human erythrocytes are capable of http://www.jbc.org/cgi/doi/10.1074/jbc.RA119.009049 The latest version is at JBC Papers in Press. Published on September 11, 2019 as Manuscript RA119.009049 by guest on June 6, 2020 http://www.jbc.org/ Downloaded from