title: Medium-chain dehydrogenases with new specificity: amino mannitol dehydrogenases on the azasugar biosynthetic pathway.
abstract: Azasugar biosynthesis involves a key dehydrogenase that oxidizes 2-amino-2-deoxy-D-mannitol to the 6-oxo compound. The genes encoding homologous NAD-dependent dehydrogenases from Bacillus amyloliquefaciens FZB42, B. atrophaeus 1942, and Paenibacillus polymyxa SC2 were codon-optimized and expressed in BL21(DE3) Escherichia coli. Relative to the two Bacillus enzymes, the enzyme from P. polymyxa proved to have superior catalytic properties with a Vmax of 0.095 ± 0.002 µmol/min/mg, 59-fold higher than the B. amyloliquefaciens enzyme. The preferred substrate is 2- amino-2-deoxy-D-mannitol, though mannitol is accepted as a poor substrate at 3% of the relative rate. Simple amino alcohols were also accepted as substrates at lower rates. Sequence alignment suggested D283 was involved in the enzyme's specificity for aminopolyols. Point mutant D283N lost its amino specificity, accepting mannitol at 45% the rate observed for 2-amino-2-deoxy-D-mannitol. These results provide the first characterization of this class of zinc-dependent medium chain dehydrogenases that utilize aminopolyol substrates.
authors.forename: Yanbin
authors.initials: Y
authors.lastname: Wu
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Arciola
authors.affiliation: Department of Chemistry, University of Florida, Gainesville Florida, 32611-7200, USA. horen@chem.ufl.edu.
authors.forename: Nicole
authors.initials: N
authors.lastname: Horenstein
mesh_terms: D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives / Q000737:chemistry; D000595:Amino Acid Sequence; D000605:Amino Alcohols / Q000737:chemistry; D000606:Amino Sugars / Q000096:biosynthesis* / Q000737:chemistry; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D008353:Mannitol / Q000737:chemistry; D008354:Mannitol Dehydrogenases / Q000235:genetics* / Q000378:metabolism; D056507:Paenibacillus / Q000201:enzymology* / Q000235:genetics; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics*; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein
chemical_list: D000605:Amino Alcohols; D000606:Amino Sugars; D001426:Bacterial Proteins; D017485:1-Deoxynojirimycin; D008353:Mannitol; D008354:Mannitol Dehydrogenases; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C013626:nojirimycin