Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 111, Issue 23, Pages E2376-E2383Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1322352111
Keywords
amino acid coevolution; genetic selection
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Funding
- National Science and Engineering Research Council of Canada
- Canadian Institutes of Health Research [MOP 97780]
- Northwest Genome Engineering Consortium
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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 non-catalytic 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 similar to 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.
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