Characterization of the AXDH gene and the encoded xylitol dehydrogenase from the dimorphic yeast Arxula adeninivorans.
The xylitol dehydrogenase-encoding Arxula adeninivorans AXDH gene was isolated and characterized. The gene includes a coding sequence of 1107 bp encoding a putative 368 amino acid protein of 40.3 kDa. The identity of the gene was confirmed by a high degree of homology of the derived amino acid sequence to that of xylitol dehydrogenases from different sources. The gene activity was regulated by carbon source. In media supplemented with xylitol, D-sorbitol and D-xylose induction of the AXDH gene and intracellular accumulation of the encoded xylitol dehydrogenase was observed. This activation pattern was confirmed by analysis of AXDH promoter-GFP gene fusions. The enzyme characteristics were analysed from isolates of native strains as well as from those of recombinant strains expressing the AXDH gene under control of the strong A. adeninivorans-derived TEF1 promoter. For both proteins, a molecular mass of ca. 80 kDa was determined corresponding to a dimeric structure, an optimum pH at 7.5 and a temperature optimum at 35 degrees C. The enzyme oxidizes polyols like xylitol and D-sorbitol whereas the reduction reaction is preferred when providing D-xylulose, D-ribulose and L-sorbose as substrates. Enzyme activity exclusively depends on NAD+ or NADH as coenzymes.
Böer
Erik
E
Institut für Pflanzengenetik und Kulturpflanzenforschung, Corrensstr. 3, D-06466 Gatersleben, Germany.
Wartmann
Thomas
T
Schmidt
Silvia
S
Bode
Rüdiger
R
Gellissen
Gerd
G
Kunze
Gotthard
G
D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D001483:Base Sequence; D050542:D-Xylulose Reductase; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004795:Enzyme Stability; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010429:Pentoses / Q000378:metabolism; D021122:Protein Subunits; D004718:Saccharomycetales / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013012:Sorbitol / Q000378:metabolism; D013013:Sorbose / Q000378:metabolism; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature; D014993:Xylitol / Q000378:metabolism; D014994:Xylose; D014996:Xylulose / Q000378:metabolism
D004271:DNA, Fungal; D010429:Pentoses; D021122:Protein Subunits; D049452:Green Fluorescent Proteins; D013012:Sorbitol; C100182:ribulose; D014996:Xylulose; D014994:Xylose; D013401:Sugar Alcohol Dehydrogenases; D050542:D-Xylulose Reductase; D013013:Sorbose; D014993:Xylitol