Directed molecular evolution of ADP-glucose pyrophosphorylase - PubMed (original) (raw)

Directed molecular evolution of ADP-glucose pyrophosphorylase

Peter R Salamone et al. Proc Natl Acad Sci U S A. 2002.

Abstract

ADP-glucose pyrophosphorylase catalyzes a rate-limiting reaction in prokaryotic glycogen and plant starch biosynthesis. Despite sharing similar molecular size and catalytic and allosteric regulatory properties, the prokaryotic and higher plant enzymes differ in higher-order protein structure. The bacterial enzyme is encoded by a single gene whose product of ca. 50,000 Da assembles into a homotetrameric structure. Although the higher plant enzyme has a similar molecular size, it is made up of a pair of large subunits and a pair of small subunits, encoded by different genes. To identify the basis for the evolution of AGPase function and quaternary structure, a potato small subunit homotetrameric mutant, TG-15, was subjected to iterations of DNA shuffling and screened for enzyme variants with up-regulated catalytic and/or regulatory properties. A glycogen selection/screening regimen of buoyant density gradient centrifugation and iodine vapor colony staining on glucose-containing media was used to increase the stringency of selection. This approach led to the isolation of a population of AGPase small subunit homotetramer enzymes with enhanced affinity toward ATP and increased sensitivity to activator and/or greater resistance to inhibition than TG-15. Several enzymes displayed a shift in effector preference from 3-phosphoglycerate to fructose-6 phosphate or fructose-1,6-bis-phosphate, effectors used by specific bacterial AGPases. Our results suggest that evolution of AGPase, with regard to quaternary structure, allosteric effector selectivity, and effector sensitivity, can occur through the introduction of a few point mutations alone with low-level recombination hastening the process.

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Figures

Figure 1

Figure 1

Selection and screening methodologies. (A) Percoll density gradient separation of glycogen-positive clones from cells unable to accumulate glycogen. (B) Cells were harvested from the upper and lower bands in A and tested for kanamycin resistance (B Upper) and glycogen accumulation by iodine staining (B Lower). (C) Representative iodine staining patterns exhibited by third-generation Devo bacterial colonies grown on 0.1% glucose: dark staining colony (black arrowhead), lightly staining colony (arrow), and negative staining colony (white arrowhead). (D) Iodine staining of selected third-generation Devo mutants on glucose-enriched media. Devo clones were streaked onto plates containing decreasing amounts of glucose and stained by exposure to iodine vapors as described in Experimental Protocols. 1, Devo 350; 2, Devo 339; 3, Devo 316; 4, Devo 301; 5, Devo 355; 6, Devo 303; 7, Devo 330; 8, heterotetrameric UpReg-1 AGPase (23), which has enhanced affinity for activator and is more resistant to inhibition than the WT enzyme. Cells expressing the WT heterotetrameric AGPase iodine stain very lightly in media containing 0.1% glucose.

Figure 2

Figure 2

Relative locations of the TG-15 and Devo mutations in the WT AGPase small subunit coding sequence. All Devo mutants retained both of the TG-15 mutations (L46F, V57I).

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