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[email protected] 10 Protein & Peptide Letters, 2014, 21, 10-14 Medium-Chain Dehydrogenases with New Specificity: Amino Mannitol Dehydrogenases on the Azasugar Biosynthetic Pathway Yanbin Wu, Jeffrey Arciola, and Nicole Horenstein* Department of Chemistry, University of Florida, Gainesville Florida, 32611-7200, USA 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 mol/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 alco- hols 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. Keywords: Aminopolyol, azasugar, biosynthesis, dehydrogenase, mannojirimycin, nojirimycin. INTRODUCTION are sufficient to convert fructose-6-phosphate into manno- jirimycin [9]. We proposed that the gutB1 gene product was Azasugars such as the nojirimycins [1] are natural prod- responsible for the turnover of 2-amino-2-deoxy-D-mannitol ucts that are analogs of monosaccharides that feature a nitro- (2AM) into mannojirimycin as shown in Fig.