title: Structural and biochemical insights into nucleotide-rhamnose synthase/epimerase-reductase from Arabidopsis thaliana.
abstract: L-Rhamnose (Rha) is synthesized via a similar enzymatic pathway in bacteria, plants and fungi. In plants, nucleotide-rhamnose synthase/epimerase-reductase (NRS/ER) catalyzes the final step in the conversion of dTDP/UDP-α-D-Glc to dTDP/UDP-β-L-Rha in an NAD(P)H dependent manner. Currently, only biochemical evidence for the function of NRS/ER has been described. In this study, a crystal structure for Arabidopsis thaliana NRS/ER was determined, which is the first report of a eukaryotic rhamnose synthase with both epimerase and reductase activities. NRS/ER functions as a metal ion independent homodimer that forms through hydrophobic interactions via a four-helix bundle. Each monomer exhibits α/β folding that can be divided into two regions, nucleotide cofactor binding domain and sugar substrate binding domain. The affinities of ligands with NRS/ER were measured using isothermal titration calorimetry, which showed that NRS/ER has a preference for dTDP over UDP, while the cofactor binding site has a similar affinity for NADH and NADPH. Structural analysis coupled to site-directed mutagenesis suggested C115 and K183 as the acid/base pair responsible for epimerization, while T113, Y144 and K148 are the conserved residues in reduction. These findings shed light on the molecular mechanism of NRS/ER and were helpful to explore other eukaryotic enzymes involved in L-Rha synthesis.
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071, China; Food and Pharmaceutical Engineering Institute, Shanxi University of Traditional Chinese Medicine, Taiyuan 030024, China. Electronic address: hxdon@126.com.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Han
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071, China; Tianjin Research Institute of Forestry and Pomology, Tianjin Academy of Agricultural Sciences, Tianjin 300192, China. Electronic address: qianlei507@126.com.
authors.forename: Lei
authors.initials: L
authors.lastname: Qian
authors.affiliation: College of Pharmacy, Collaborative Innovation Center for Biotherapy, and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, China. Electronic address: lianwen@nankai.edu.cn.
authors.forename: Lianwen
authors.initials: L
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071, China. Electronic address: liu2008@nankai.edu.cn.
authors.forename: Xinqi
authors.initials: X
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry* / Q000201:enzymology; D001665:Binding Sites; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry*; D009249:NADP / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012210:Rhamnose / Q000096:biosynthesis / Q000737:chemistry*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D011994:Recombinant Proteins; D009243:NAD; D009249:NADP; D002238:Carbohydrate Epimerases; C488385:dTDP-rhamnose synthase, Arabidopsis; D012210:Rhamnose
keywords: Active site; Arabidopsis thaliana; Crystal structure; Nucleotide–rhamnose synthase/epimerase–reductase