Structure‐Function Studies of Deinococcus radiodurans ADP Glucose Pyrophosphorylase: Role of Ser48 in Allosteric Regulation

Abstract

Adenosine Diphosphate Glucose Pyrophosphorylase (ADPG PPase) is an allosterically regulated enzyme that functions 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 in crops. The microbial versions of this enzyme are quite diverse in their regulatory and physical properties; some of these properties would be useful to incorporate into transgenic crops to enhance starch production. While a number of ADPG PPases have been kinetically characterized, there is only one published x ray structure (PDB 3BRK) of an inhibited form. Very little is known about the enzyme from Deinococcus radiodurans (D. rad), an extremophile that is resistant to ionizing radiation and harsh growth conditions. When comparing the amino acid sequence of this enzyme to other characterized ADPG PPases, it was noted that position 48 differed with a serine substituted for alanine in a region known to be important for allostery. To probe the role of Ser‐48, the S48A enzyme was generated by site‐directed mutagenesis and the recombinant altered D. rad ADPG PPases successfully expressed in E. coli and purified via a scheme that includes anion exchange chromatography, size exclusion chromatography, and affinity chromatography. Initial studies on the S48A enzyme in the absence of activators have shown a dramatic 20‐fold increase in the apparent affinity for the substrate ATP, a 3‐fold increase in apparent affinity for the cofactor magnesium, and a 4‐fold increase in Vmax compared to wild‐type. Interestingly, in the presence of the activator FBP there was little change in the apparent binding affinity for substrates or Vmax compared to WT which displays a 14‐fold increase in Vmax and 12‐fold and 3‐fold increase in apparent affinity for ATP and magnesium, respectively. Similarly, in the presence of F6P, there were not a very large increase in Vmax or higher apparent affinity for ATP for the S48A enzyme, but there was a 9‐fold difference in Vmax and a 12‐fold and 2‐fold increase in apparent binding affinity for ATP and magnesium for WT, respectively. The WT enzyme also displayed higher apparent affinity for FBP and F6P compared to the S48A enzyme. The alanine substitution appears to result in an enzyme form that is partially activated but relatively insensitive to activators. Further kinetic characterization of S48A in the presence of sulfate and the inhibitor phosphate is in progress. With respect to physical characterization, two crystallization conditions for S48A, one including imidazole and the other with lithium sulfate and PEG, were found to yield preliminary results. Further crystallization trials are in progress as a first step in elucidating the three‐dimensional structure.Support or Funding InformationSupported in part by NSF and NSF BIO MCB grant #0448676.