P54 Isolation of levoglucosan-utilizing bacteria and crystallographic characterization of levoglucosan dehydrogenase
Monday, July 25, 2016
Grand Ballroom, 5th Fl (Sheraton New Orleans)
A. Arya* and M.A. Eiteman, University of Georgia, Athens, GA
Pyrolysis of lignocellulose generates syngas, bio-oil, and bio-char useful as fuels.  The process also generates high yields of levoglucosan.  Because this sugar is not readily metabolized by typical production organisms such as E. coli and S. cerevisiae, there has been interest in identifying enzymes which metabolize levoglucosan and engineering production organisms with these enzymes.

            Levoglucosan dehydrogenase (LGDH) is the only known bacterial enzyme to act on levoglucosan, and previously only one species (Arthrobacter) is known to express LGDH.  LGDH is proposed to oxidize levoglucosan to 3-keto-levoglucosan, which then chemically hydrates to 3-keto-glucose before LGDH reduces this substrate to glucose.  Is this enzyme and this metabolic pathway specific to one taxonomic group of bacteria?  What residues make LGDH specific to levoglucosan conversion?

            We isolated five bacteria showing growth on levoglucosan and LGDH activity.  Illumina sequencing identified these isolates to be from the genera Microbacterium, Paenibacillus, Rhizobium, and Klebsiella.  The gene lgdh has been identified in four isolates, and we are presently looking at neighboring genes for other enzymes critical to prokaryotic levoglucosan metabolism.

            Additionally, to understand how LGDH interacts with levoglucosan, the Arthrobacter LGDH was crystallized.  X-ray diffraction data were used to assemble enzyme structures, revealing that while major structural features including the Rossman-fold for NADH binding and the carbohydrate active site are conserved relative to known dehydrogenases, LGDH has several different residues in the carbohydrate-binding site likely responsible for its preferential activity on levoglucosan.  This information suggests how the enzyme might be engineered for improved levoglucosan degradation.