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abstracts
Lignocellulosic biomass is an abundant source of carbohydrates that can be used for the production of biofuels. Additional processing of biomass-derived sugars arises from the fact that the most abundant sugar product from biomass pyrolysis is the unusual anhydro-ring containing sugar, levoglucosan (LG). LG does not fall within the native substrate range of commonly used biocatalysts such as Escherichia coli and Saccharomyces cerevisiae. However, LG can be further converted to glucose-6-phosphate through the activity of the sugar kinase known as levoglucosan kinase (LGK), which has been isolated from a variety of fungal sources. Integration of recombinant LGK from Lipomyces starkeyi into an engineered ethanologenic Escherichia coli strain has been shown to allow for the utilization of LG as the sole carbon source for ethanol production [1]. However, challenges associated with effective utilization of LG include a high Km of LGK for LG, and the relatively low activity of LGK at physiological pH. In addition to the practical applications of LGK for biofuel production, the enzyme is an exceptional target for structural and mechanistic studies since it appears to possess dual hydrolase and kinase functionality. In order to gain a better understanding of the structure and mechanism of LGK, we have crystallized and determined several high-resolution X-ray structures of Lipomyces starkeyi LGK bound to reaction substrates and products. We have also recently collected low-resolution neutron diffraction for an LGK crystal, and further optimization of LGK crystals is currently underway to improve crystal size and diffraction. Neutron diffraction will reveal the protonation states of key residues in the active site of LGK and provide highly detailed information about hydrogen bonds, including water-bonding interactions. The rational design of new LGK constructs will be used to improve applications of this enzyme towards levoglucosan derived biofuel production.
abstracts
Protein ubiquitination regulates important innate immune responses. Ubiquitin (Ub) can be attached to lysine residues on cellular proteins to promote, among other activities, the innate immune responses of the cell. These pathways can in turn be downregulated by the removal of Ub from cellular proteins by deubiquitinases (DUBs). Viruses of the order Nidovirales have positive-sense, single stranded RNA genomes. Within this order are the families Coronaviridae and Arteriviridae, which include viruses known to cause severe disease in humans and animals, respectively. Members of the families Coronaviridae and Arteriviridae share a common mechanism of gene expression, whereby the viral nonstructural proteins (nsps) are initially expressed as a single polyprotein, which is then cleaved into functional units by papain-like protease (PLP) domains encoded within. Interestingly, while also being necessary for viral replication, a number of Nidovirus PLPs have been shown to remove Ub from host proteins, in order to down-regulate the host innate immune response. Here we present the crystal structure of a Nidovirus PLP in complex with Ub. The structure allowed for the characterization of a Ub-binding interface, and identification of specific residues involved in Ub recognition that are distant from the enzyme active site. The selective inactivation of DUB activity of viral PLP enzymes verses their polyprotein cleavage activity by site directed mutagenesis is allowing us to understand the role of DUB activity in evading innate immune responses of the host, and opens the door for the development of improved live attenuated vaccines against Nidoviruses and other viruses encoding similar dual specificity proteases.
