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Abstract
The gene for levoglucosan dehydrogenase, lgdA, was identified in Pseudarthrobacter phenanthrenivorans Sphe3 by searching the genome with known peptide sequences. The bacteria Paenibacillus athensensis MEC069T, Shinella sumterensis MEC087T, Microbacterium marinilacus MEC084, and Klebsiella pneumoniae MEC097 were isolated from soil and wastewater based on their ability to consume levoglucosan, and the genomes of these organisms were assembled and screened for lgdA. Identified lgdA were recombinantly expressed in E. coli BL21 (DE3)-RIPL cells, and the levoglucosan dehydrogenases (LGDH) were purified by FPLC and kinetically characterized based on their activity in the presence of levoglucosan and NAD+. These enzymes were found to vary significantly. LGDH from P. phenanthrenivorans and K. pneumoniae showed a KM for levoglucosan of 27 and 22 mM, respectively, and both enzymes showed no sign of substrate inhibition. The LGDH from P. athensensis, S. sumterensis, and M. marinilacus MEC084 showed KM for levoglucosan of ~2 mM and were inhibited by levoglucosan at concentrations as low as 93 mM. All LGDH showed KM for NAD+ in the 0.3-1.0 mM range, except for the the enzyme from S. sumterensis with a KM of 9.1 mM. Purified P. phenanthrenivorans Sphe3 LGDH was crystallized, and some crystals were soaked with the substrates, levoglucosan or glucose, and NADH. The X-ray structures were solved by molecular replacement to resolve which residues make up the active site and how substrates bind to the active site. Based on the observed structures, the mechanism of catalysis in LGDH was proposed to be dependent on a His-Lys-Asp triad conserved in similar oxidoreducatases, in which His begins catalysis by taking the proton from hydroxyl-3 of levoglucosan and a hydride is shifted from levoglucosan C3 to the nicotinamide ring C4. Lys and Asp are likely to form a proton relay to remove the proton from His so that catalysis may continue.