title: Transcriptome-guided gene isolation and functional characterization of UDP-xylose synthase and UDP-D-apiose/UDP-D-xylose synthase families from Ornithogalum caudatum Ait.
abstract: The present study first identified the involvement of OcUAXS2 and OcUXS1-3 in anticancer polysaccharides biosynthesis in O. caudatum. UDP-xylose synthase (UXS) and UDP-D-apiose/UDP-D-xylose synthase (UAXS), both capable of converting UDP-D-glucuronic acid to UDP-D-xylose, are believed to transfer xylosyl residue to anticancer polysaccharides biosynthesis in Ornithogalum caudatum Ait. However, the cDNA isolation and functional characterization of genes encoding the two enzymes from O. caudatum has never been documented. Previously, the transcriptome sequencing of O. caudatum was performed in our laboratory. In this study, a total of six and two unigenes encoding UXS and UAXS were first retrieved based on RNA-Seq data. The eight putative genes were then successfully isolated from transcriptome of O. caudatum by reverse transcription polymerase chain reaction (RT-PCR). Phylogenetic analysis revealed the six putative UXS isoforms can be classified into three types, one soluble and two distinct putative membrane-bound. Moreover, the two UAXS isoenzymes were predicted to be soluble forms. Subsequently, these candidate cDNAs were characterized to be bona fide genes by functional expression in Escherichia coli individually. Although UXS and UAXS catalyzed the same reaction, their biochemical properties varied significantly. It is worth noting that a ratio switch of UDP-D-xylose/UDP-D-apiose for UAXS was established, which is assumed to be helpful for its biotechnological application. Furthermore, a series of mutants were generated to test the function of NAD+binding motif GxxGxxG. Most importantly, the present study determined the involvement of OcUAXS2 and OcUXS1-3 in xylose-containing polysaccharides biosynthesis in O. caudatum. These data provide a comprehensive knowledge for UXS and UAXS families in plants.
authors: State Key Laboratory of Bioactive Substance and Function of Natural Medicines and Ministry of Health Key Laboratory of Biosynthesis of Natural Products, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
authors: Sen
authors: S
authors: Yin
authors: State Key Laboratory of Bioactive Substance and Function of Natural Medicines and Ministry of Health Key Laboratory of Biosynthesis of Natural Products, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. jianqiangk@imm.ac.cn.
authors: Jian-Qiang
authors: JQ
authors: Kong
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D064751:Ammonium Compounds / Q000494:pharmacology; D055162:Biocatalysis / Q000187:drug effects; D002021:Buffers; D002118:Calcium / Q000494:pharmacology; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D017343:Genes, Plant; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D005810:Multigene Family; D009928:Organ Specificity / Q000187:drug effects / Q000235:genetics; D031442:Ornithogalum / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D066244:Proton Magnetic Resonance Spectroscopy; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013696:Temperature; D059467:Transcriptome / Q000187:drug effects / Q000235:genetics*; D014539:Uridine Diphosphate Sugars / Q000737:chemistry / Q000378:metabolism*; D014540:Uridine Diphosphate Xylose / Q000737:chemistry / Q000378:metabolism*
chemical_list: D064751:Ammonium Compounds; D002021:Buffers; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; C511616:UDP-apiose; D014539:Uridine Diphosphate Sugars; D014540:Uridine Diphosphate Xylose; D002262:Carboxy-Lyases; C023257:UDPglucuronate decarboxylase; D002118:Calcium
keywords: Ornithogalum caudatum; Transcriptome sequencing; UDP-D-apiose/UDP-D-xylose synthase; UDP-D-xylose synthase; Xylose-containing polysaccharides