Sunday, August 12, 2012
Columbia Hall, Terrace Level (Washington Hilton)
Levoglucosan (1,6-anhydro-β-D-glucopyranose) is produced from the pyrolysis of naturally abundant carbohydrates such as cellulose and starch, and consequently, is a major product of biomass pyrolysis. Lipomyces starkeyi levoglucosan kinase (LGK) belongs to the hexokinase family of sugar kinases and is known to convert the anhydrosugar to glucose-6-phosphate in an ATP-dependent reaction. While bacteria such as E. coli lack the ability to metabolize levoglucosan, it has recently been shown that genomic integration of a codon-optimized LGK from Lipomyces starkeyi into an engineered ethanologenic E. coli strain allows for the utilization of levoglucosan as a carbon source for the production of ethanol. LGK shows sequence homology to the functionally related 1,6-anhydro-N-acetylmuramic acid kinase known as AnmK that is encoded by many pathogenic Gram-negative bacteria. We have previously determined crystal structures of AnmK and proposed a mechanism of catalysis, whereby a catalytic enzymatic aspartate residue acts as a base to activate a water molecule, which then attacks the substrate anomeric carbon, allowing for cleavage of the anhydro bond and phosphorylation of the liberated O6 oxygen. We have now also purified and crystallized the codon-optimized LGK from Lipomyces starkeyi and are investigating the function of this enzyme in conjunction with our studies of AnmK. A detailed understanding of the structure and mechanism of LGK will assist in the rational design of mutations that will be targeted to enhance the specificity, rate of catalysis and stability of this enzyme, which will have downstream practical applications for levoglucosan derived biofuel production.