title: L-arabinose/D-galactose 1-dehydrogenase of Rhizobium leguminosarum bv. trifolii characterised and applied for bioconversion of L-arabinose to L-arabonate with Saccharomyces cerevisiae.
abstract: Four potential dehydrogenases identified through literature and bioinformatic searches were tested for L-arabonate production from L-arabinose in the yeast Saccharomyces cerevisiae. The most efficient enzyme, annotated as a D-galactose 1-dehydrogenase from the pea root nodule bacterium Rhizobium leguminosarum bv. trifolii, was purified from S. cerevisiae as a homodimeric protein and characterised. We named the enzyme as a L-arabinose/D-galactose 1-dehydrogenase (EC 1.1.1.-), Rl AraDH. It belongs to the Gfo/Idh/MocA protein family, prefers NADP(+) but uses also NAD(+) as a cofactor, and showed highest catalytic efficiency (k cat/K m) towards L-arabinose, D-galactose and D-fucose. Based on nuclear magnetic resonance (NMR) and modelling studies, the enzyme prefers the α-pyranose form of L-arabinose, and the stable oxidation product detected is L-arabino-1,4-lactone which can, however, open slowly at neutral pH to a linear L-arabonate form. The pH optimum for the enzyme was pH 9, but use of a yeast-in-vivo-like buffer at pH 6.8 indicated that good catalytic efficiency could still be expected in vivo. Expression of the Rl AraDH dehydrogenase in S. cerevisiae, together with the galactose permease Gal2 for L-arabinose uptake, resulted in production of 18 g of L-arabonate per litre, at a rate of 248 mg of L-arabonate per litre per hour, with 86 % of the provided L-arabinose converted to L-arabonate. Expression of a lactonase-encoding gene from Caulobacter crescentus was not necessary for L-arabonate production in yeast.
authors.affiliation: VTT, Technical Research Centre of Finland, P.O. Box 1000, 02044, Espoo, Finland.
authors.forename: Niina
authors.initials: N
authors.lastname: Aro-Kärkkäinen
authors.forename: Mervi
authors.initials: M
authors.lastname: Toivari
authors.forename: Hannu
authors.initials: H
authors.lastname: Maaheimo
authors.forename: Mikko
authors.initials: M
authors.lastname: Ylilauri
authors.forename: Olli T
authors.initials: OT
authors.lastname: Pentikäinen
authors.forename: Martina
authors.initials: M
authors.lastname: Andberg
authors.forename: Merja
authors.initials: M
authors.lastname: Oja
authors.forename: Merja
authors.initials: M
authors.lastname: Penttilä
authors.forename: Marilyn G
authors.initials: MG
authors.lastname: Wiebe
authors.forename: Laura
authors.initials: L
authors.lastname: Ruohonen
authors.forename: Anu
authors.initials: A
authors.lastname: Koivula
mesh_terms: D001089:Arabinose / Q000378:metabolism*; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D004795:Enzyme Stability; D005691:Galactose Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016961:Rhizobium leguminosarum / Q000201:enzymology* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013400:Sugar Acids / Q000378:metabolism*
chemical_list: D003067:Coenzymes; D011994:Recombinant Proteins; D013400:Sugar Acids; D009243:NAD; D009249:NADP; D001089:Arabinose; D005691:Galactose Dehydrogenases; C047959:galactose dehydrogenase