Glucans serve as critical energy storage compounds in nearly all living organisms. Such renewable and biodegradable carbon sources are in demand now from industry as substitutes for petroleum based products. ADP-Glucose Pyrophosphorylase (ADPG PPase) is the enzyme that catalyzes the rate limiting step of glucan biosynthesis in plants and bacteria, respectively. The novel Thermodesulfovibrio yellowstonii (Td.y ) ADPG PPase, from a thermophilic, sulfate-reducing bacteria, has potential properties that could lead to engineering a very stable and highly active form which could increase biomass yield. Further, sequence alignment data indicate that Td.y harbors varied amino acids in two conserved N-terminal and C-terminal regions that are known to be important for regulation in other characterized ADPG PPases which may be responsible for its different effector specificity that includes activation by PEP. To probe the functional roles of these regions, the variants E15A, F23R, S25A, K365G, R397A were generated and the recombinant proteins purified for kinetic analyses. For E15A, the kinetic parameters were similar to WT in the absence of effectors but in the presence of PEP shows two-fold higher activation than WT. In the absence of effector molecules, data from F23R reveal a 4 fold decrease in activity and a ~2 fold decrease in apparent affinity for the substrate ATP compared to WT. However, in the presence of PEP, ATP affinity was restored to WT values. Kinetic data for the variant S25 reveals a Vmax ~3 fold higher than WT with a10-fold higher apparent affinity for ATP as well as desensitization to effector molecules. The K365G enzyme displayed two fold lower activity than WT and substantial desensitization to PEP. The R397A enzyme exhibited a ~20 fold reduction in activity compared to WT and inhibition by PEP. Along with kinetic analyses, initial crystallization trials are also underway. Results from the WT and variants confirm and extend the roles of the conserved regions for activity, regulation, and allosteric specicity. This material is based upon work supported by the National Science Foundation (in part by #0448676).

Probing the role of N and C-terminal regions in allosteric regulation of <i>Thermodesulfovibrio yellowstonii </i>ADP-glucose pyrophosphorylase

251st National Meeting (2016)

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Probing the role of N and C-terminal regions in allosteric regulation of Thermodesulfovibrio yellowstonii ADP-glucose pyrophosphorylase