Structure/function studies of Diverse Forms of ADPGlucose Pyrophosphorylase

Abstract

ADPGlucose Pyrophosphorylase (ADPG PPase) is an allosterically regulated enzyme that serves as the rate‐limiting step of starch synthesis in plants and glycogen synthesis in bacteria. Because starch is a source of renewable and biodegradable carbon, ADPG PPase is an attractive target for protein engineering to increase biomass yield. The microbial versions of this enzyme are quite diverse in their regulatory and physical properties in accord with adaptation to different environments; some of these properties would be useful to incorporate into crops to enhance starch production.The enzyme from Deinococcus radiodurans (D. rad), coded for by the glgC gene, is an extremophile resistant to radiation and harsh growth conditions. Using standard assays with magnesium (Mg) as a co‐factor, the purified D. rad enzyme was found to have relatively low apparent affinity for the substrate ATP and Mg with S0.5 values of 8.4 mM and 24 mM, respectively. However, this organism is known to have a high intracellular manganese (Mn) concentration, which correlates with ionizing radiation and desiccation resistance. Because stored glycogen could act as an energy and carbon source during cell recovery, it was hypothesized that this ADPG PPase may be more responsive to Mn. Use of Mn in the assay revealed a ~9.3 fold increase in apparent affinity for ATP, a ~5 fold increase in apparent affinity for Mn (versus Mg), and a two‐fold increase in Vmax which supports the hypothesis. Complete kinetic characterization of the wild‐type (WT) and selected altered enzymes is underway to probe the putative metal binding site(s). Several crystallization conditions were found to yield promising results and further crystallization trials are in progress.In contrast, the enzyme from the thermophile Thermotoga maritima (T.ma) consists of two subunits, coded for by the glgC and glgD genes. Previous studies indicated that the purified WT T.ma glgD alone had no activity but was found to stimulate the relatively low activity of the glgC subunit. The complex could also be activated by FBP while the glgC alone was insensitive to FBP. The apparent affinity for ATP and Mg increased ~2 and ~3 fold in the complex while the Vmax increased 4 fold. Previous results indicated that limited proteolysis of the C‐terminus of glgD resulted in lower activation of the complex. In order to probe this further, a 73 amino acid C‐terminal truncation was generated and the activity of the complexes with the WT and WT glgC/truncated glgD (altered complex) compared. The altered complex exhibited a lower apparent affinity for ATP than the WT complex as well as a 5‐fold reduction in Vmax, which still represented a ~3‐fold stimulation compared to the glgC alone. In the presence of the activator FBP, the altered complex was slightly inhibited by FBP in contrast to the 2‐fold activation of the WT complex. The results appear to support a role for the C‐terminus in allosteric activation by FBP and partial stimulation of the enzyme in the absence of FBP. A 3 Å crystal structure for the glgC subunit was recently solved which was in good agreement with a previously modeled structure based on the A. tumefaciens enzyme structure (PDB 3BRK). Complete kinetic and physical characterization of the altered enzyme complexes in the absence and presence of FBP, as well as crystallization of various complexes are underway.Support or Funding InformationSupported in part by NSF BIO MCB Grant 0448676 and BIO DBI.