Rational engineering of enzyme allosteric regulation through sequence evolution analysis

• Published in: PLoS computational biology 2012-07, Vol.8 (7), p.e1002612-e1002612
• Main Authors: Yang, Jae-Seong, Seo, Sang Woo, Jang, Sungho, Jung, Gyoo Yeol, Kim, Sanguk
• Format: Article
• Language: English
• Subjects: Adenosine Monophosphate - chemistry; Adenosine Monophosphate - metabolism; Allosteric proteins; Allosteric Regulation; Amino Acid Sequence; Binding sites; Biology; Catalytic Domain; Computational Biology; DNA sequencing; Engineering; Enzymes; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Evolution, Molecular; Fructose-Bisphosphatase - chemistry; Fructose-Bisphosphatase - genetics; Fructose-Bisphosphatase - metabolism; Genetic engineering; Genetic regulation; Glucose-6-Phosphate - chemistry; Glucose-6-Phosphate - metabolism; Hydrophobic and Hydrophilic Interactions; Kinases; Models, Molecular; Molecular Sequence Data; Mutation; Nucleotide sequencing; Physiological aspects; Protein Engineering - methods; Proteins; Sequence Analysis, Protein - methods
• ISSN: 1553-7358; 1553-734X; 1553-7358
• DOI: 10.1371/journal.pcbi.1002612

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.