Probing the Role of Ser‐48 in the Regulation of Deinococcus radiodurans ADP Glucose 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; some of these properties would be useful to incorporate into crops to enhance starch production. Little is known about the enzyme from Deinococcus radiodurans (D. rad), an extremophile resistant to radiation and harsh growth conditions. When comparing the sequence of this enzyme to others, position 48 differed with a serine substituted for alanine in a region known to be important for allostery. To probe the role of S48, the S48A enzyme was generated by site-directed mutagenesis and the altered D. rad ADPG PPase successfully expressed and purified. Initial studies on the S48A enzyme in the absence of activators have shown a 20-fold increase in the apparent affinity for the substrate ATP and a 4-fold increase in Vmax compared to wild-type. Interestingly, in the presence of the activators FBP and F6P there were no change in the apparent binding affinity for substrates or Vmax compared to WT which displays a 5-fold increase in Vmax and 12-fold and 3-fold increase in apparent affinity for ATP and magnesium, respectively. The alanine substitution appears to result in an enzyme form that is partially activated but insensitive to activators. Complete kinetic analyses of the S48A enzyme are in progress. Supported in part by NSF Grant 0448676.