Directed molecular evolution of ADP-glucose pyrophosphorylase.
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.
Salamone
Peter R
PR
Washington State University, Institute of Biological Chemistry, School of Molecular Biosciences, Pullman, WA 99164-6340, USA.
Kavakli
I Halil
IH
Slattery
Casey J
CJ
Okita
Thomas W
TW
D019020:Directed Molecular Evolution / Q000379:methods; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D017343:Genes, Plant; D051599:Glucose-1-Phosphate Adenylyltransferase; D007700:Kinetics; D009154:Mutation; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D021122:Protein Subunits; D011198:Solanum tuberosum / Q000201:enzymology / Q000235:genetics
D021122:Protein Subunits; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase