Rational engineering of enzyme allosteric regulation through sequence evolution analysis.
Control of enzyme allosteric regulation is required to drive metabolic flux toward desired levels. Although the three-dimensional (3D) structures of many enzyme-ligand complexes are available, it is still difficult to rationally engineer an allosterically regulatable enzyme without decreasing its catalytic activity. Here, we describe an effective strategy to deregulate the allosteric inhibition of enzymes based on the molecular evolution and physicochemical characteristics of allosteric ligand-binding sites. We found that allosteric sites are evolutionarily variable and comprised of more hydrophobic residues than catalytic sites. We applied our findings to design mutations in selected target residues that deregulate the allosteric activity of fructose-1,6-bisphosphatase (FBPase). Specifically, charged amino acids at less conserved positions were substituted with hydrophobic or neutral amino acids with similar sizes. The engineered proteins successfully diminished the allosteric inhibition of E. coli FBPase without affecting its catalytic efficiency. We expect that our method will aid the rational design of enzyme allosteric regulation strategies and facilitate the control of metabolic flux.
School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Gyeongbuk, Korea.
Jae-Seong
JS
Yang
Sang Woo
SW
Seo
Sungho
S
Jang
Gyoo Yeol
GY
Jung
Sanguk
S
Kim
D000249:Adenosine Monophosphate / Q000737:chemistry / Q000378:metabolism; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D019295:Computational Biology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D006597:Fructose-Bisphosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000737:chemistry / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D020539:Sequence Analysis, Protein / Q000379:methods*
D029968:Escherichia coli Proteins; D000249:Adenosine Monophosphate; D019298:Glucose-6-Phosphate; D006597:Fructose-Bisphosphatase