Endogenous Synthesis of Erythritol, a Novel Biomarker of Weight Gain (P15-016-19)

Abstract

ObjectivesSerum erythritol is associated with central adiposity gain in young adults. Erythritol, a 4-carbon polyol, is synthesized endogenously from erythrose through the pentose phosphate pathway. We have identified two enzymes which catalyze this reaction: alcohol dehydrogenase 1 (ADH1) and sorbitol dehydrogenase (SORD). Interestingly, ADH1 isoforms ADH1B1 and ADH1C2 catalyze NADPH-dependent synthesis of erythritol in vitro, but ADH1B1 does not. In A549 cells, siRNA knockdown of SORD levels to less than 15% of control levels reduced erythritol by 50%. A549 cells also have low levels of ADH1 expression. This indicates that other enzymes may be capable of endogenous erythritol production. Based on its high degree of homology to ADH1, we hypothesize that ADH4 also catalyzes erythritol synthesis. The objective of this study was to further elucidate the mechanism of erythritol synthesis by: determining key differences between the active site of ADH1B2 compared to ADH1B1 and SORD, and screening a new candidate enzyme, ADH4. MethodsThis study used molecular modeling to evaluate the enzyme, erythrose, NADPH complex. Site-directed mutagenesis was performed to replace Arg208 (R208) of recombinant human SORD with histidine. Recombinant SORD R208H mutant and ADH4 were then expressed, purified, and their erythrose reduction activity was kinetically characterized. ResultsMolecular modeling of the active sites of ADH1B1 and ADH1B2 revealed that the Arg48 residue of ADH1B1 makes three contacts with NADPH, whereas His48 of ADH1B2 only makes one contact. Mutation of the SORD active-site Arg208 to histidine reduced the kcat by more than 80% compared to wild-type (95 ± 21 min−1 wild-type, 16 ± 2 min−1 R208H, P < 0.05). ADH4 erythrose reduction activity reduced by more than 95% compared to ADH1 variant proteins in vitro (kcat 2 ± 1 min−1). ConclusionsMutation of arginine 208 to histidine in the active site of SORD significantly reduced the kcat, indicating that the presence of an arginine residue is essential for binding NADPH, the cofactor required in vivo for erythritol synthesis. The low erythrose reduction activity of ADH4 suggests that ADH4 does not contribute to erythritol synthesis in humans. Funding SourcesNone.