title: Control of catalytic efficiency by a coevolving network of catalytic and noncatalytic residues.
abstract: The active sites of enzymes consist of residues necessary for catalysis and structurally important noncatalytic residues that together maintain the architecture and function of the active site. Examples of evolutionary interactions between catalytic and noncatalytic residues have been difficult to define and experimentally validate due to a general intolerance of these residues to substitution. Here, using computational methods to predict coevolving residues, we identify a network of positions consisting of two catalytic metal-binding residues and two adjacent noncatalytic residues in LAGLIDADG homing endonucleases (LHEs). Distinct combinations of the four residues in the network map to distinct LHE subfamilies, with a striking distribution of the metal-binding Asp (D) and Glu (E) residues. Mutation of these four positions in three LHEs--I-LtrI, I-OnuI, and I-HjeMI--indicate that the combinations of residues tolerated are specific to each enzyme. Kinetic analyses under single-turnover conditions revealed that I-LtrI activity could be modulated over an ∼100-fold range by mutation of residues in the coevolving network. I-LtrI catalytic site variants with low activity could be rescued by compensatory mutations at adjacent noncatalytic sites that restore an optimal coevolving network and vice versa. Our results demonstrate that LHE activity is constrained by an evolutionary barrier of residues with strong context-dependent effects. Creation of optimal coevolving active-site networks is therefore an important consideration in engineering of LHEs and other enzymes.
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada N6A 5C1.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: McMurrough
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada N6A 5C1.
authors.forename: Russell J
authors.initials: RJ
authors.lastname: Dickson
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada N6A 5C1.
authors.forename: Stephanie M F
authors.initials: SM
authors.lastname: Thibert
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada N6A 5C1 dedgell@uwo.ca ggloor@uwo.ca.
authors.forename: Gregory B
authors.initials: GB
authors.lastname: Gloor
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, ON, Canada N6A 5C1 dedgell@uwo.ca ggloor@uwo.ca.
authors.forename: David R
authors.initials: DR
authors.lastname: Edgell
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis; D020134:Catalytic Domain / Q000235:genetics*; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019143:Evolution, Molecular; D018698:Glutamic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008957:Models, Genetic; D008958:Models, Molecular; D009154:Mutation; D010802:Phylogeny; D017434:Protein Structure, Tertiary
chemical_list: D001224:Aspartic Acid; D018698:Glutamic Acid; D004720:Endonucleases
keywords: amino acid coevolution; genetic selection