Structure‐Function Studies of the Thermotoga maritima ADPGlucose Pyrophosphorylase: Probing the role of the C‐terminus of the GlgD subunit

Abstract

ADP Glucose pyrophosphorylase (ADPG PPase) catalyzes the rate‐limiting step of glycogen and starch biosynthesis in plants and bacteria. Structure‐function studies of the enzyme from different sources will allow the engineering of a more active enzyme to produce more renewable and biodegradable carbon. In Thermotoga maritima (T.ma) there are two genes, glgC and glgD, which code for two subunits of this ADPG PPase. The recombinant His‐tagged subunits were successfully purified using a heat step and nickel chromatography. Previous studies performed at 37ºC (pH 7.5) had shown that the wild type (WT) glgD alone had no activity, while the glgC protein displayed a specific activity of ~ 3 Unit/mg with S0.5 values for ATP and magnesium (Mg2+) of 5.39 mM and 18.66 mM, respectively. However, the WT glgC/D complex at subsaturating substrate conditions was stimulated ~20‐fold compared to glgC alone and was also activated ~2‐fold by fructose‐1,6‐ bisphosphate (FBP). The apparent affinity for ATP and Mg increased ~2 and ~3 fold in the complex while the Vmax increased 4 fold. In the presence of FBP, the apparent affinity for ATP increased ~6‐fold for the complex with a modest ~30% increase in Vmax. Previous alignment studies and molecular modeling principally focused on the functional role of the N‐terminus. Therefore the truncation of 73 amino acids at the C‐terminal end of glgD was generated to test its potential functional role in the complex. In the absence of FBP, the truncated complex exhibited a modest decrease in ATP apparent affinity for ATP and Mg2+ with ~20‐fold and ~2 fold decreased in Vmax, respectively, compared to the WT complex. It was noted that in the presence of the truncated glgD, the Vmax was substantially lower than for glgC alone, indicating an inhibitory role. In the presence of FBP, the truncated complex displayed a ~6‐fold decrease in apparent affinity for ATP, ~3‐fold for Mg and a ~6‐fold lower Vmax compared to the WT complex. However, the Vmax of the truncated complex is close to the Vmax of the glgC alone indicating that FBP diminishes the inhibition by the truncated glgD. The primary effect of the truncation appears to be on the Vmax of the complex as well as a reduction in the K‐type activation by FBP observed with the WT. Complete kinetic and physical characterization of the altered enzyme complexes in the absence and presence of FBP, as well as crystallization of WT GlgC/WT GlgD are still underway.Support or Funding InformationSupported in part by NSF and NSF BIO MCB grant #0448676.