Probing the role of the N‐terminus in the ADP‐Glucose Pyrophosphorylase from Thermodesulfovibrio yellowstonii

• DOI: 10.1096/fasebj.30.1_supplement.835.4
• Publication Date: 2021-06-08T00:57:12Z
• Authors: Eric Yik; Edward Pushkarev; Dylan Campbell; Casey Diep; Nicholas Duran; Jackson Nguyen; Andrew Orry; Christopher R Meyer
• Keywords: 0303 health sciences; 03 medical and health sciences

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 an eco‐friendly substitute 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 bacterium, has potential properties that could lead to engineering a very stable and highly active form which could increase the yield of starch. Further, sequence alignment data indicate that Td.y harbors varied amino acids in two conserved N‐terminal regions (glycine rich and RRAKPAV) that are known to be important for regulation and catalysis in other characterized ADPG PPases. The recombinant Wild‐type (WT) and variants (E15A, F23R, S25A) have been successfully purified by hydroxyapatite and size exclusion chromatography. In the case of E15A, preliminary data displayed similar velocity and similar apparent substrate affinity for ATP compared to WT, in the absence of effector molecules. However, in the presence of the activator PEP, E15A shows a ~7 fold increase in activity compared to 4 fold in WT. This suggests a role in regulation and binding of effector molecules. In the absence of effector molecules, preliminary data from F23R reveal a 4 fold decrease in activity and a ~2 fold decrease in apparent affinity for ATP compared to the WT. However, in the presence of effector molecules, ATP affinity was restored to WT values. In the presence of PEP and FBP, F23R displays a ~7 fold increase in activation compared to WT. The F23 position appears to play a role in structure or folding/allosteric regulation, which is also supported by molecular modeling. These results suggest a potential for altered binding of negatively charged metabolites due to increased positively charged residues in the postulated binding pocket. Kinetic data for the variant S25A reveals a Vmax ~3 fold higher than WT with a10‐fold higher apparent affinity for ATP compared to the WT as well as desensitization to effector molecules. This position in Td.y may play a role in setting the relative activity level that can be further attenuated by effectors. Along with kinetic analyses, initial crystallization trials are underway for variant S25A with initial crystals from different conditions diffracting to a resolution of ~3.96 Å. Results gathered from the WT and variants thus far confirm and extend the role of the two conserved regions for activity and regulation.Support or Funding InformationSupported in part by NSF and NSF BIO MCB grant #0448676.