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.amino acid coevolution | genetic selection T he active sites of enzymes are often the most conserved positions in a multiple sequence alignment, as purifying selection for maintenance of function constrains amino acid variation of residues that directly participate in catalysis. Noncatalytic residues, often in close proximity to catalytic residues, contribute to enzymatic function by maintaining the architecture and chemical environment of the active site. Noncatalytic residues often show sequence variation in multiple sequence alignments, yet nonpermissive substitutions at these positions will have an impact on enzymatic function by disrupting the architecture or chemical environment necessary for catalysis. Thus, catalytic and noncatalytic residues must coevolve with each other and surrounding residues to maintain active-site conformation and chemistry and to buffer against potentially deleterious mutations (1, 2). Coevolving residues within a protein family can be predicted by computational methods that use mutual information theory to identify residue covariation in a multiple sequence alignment (2-5). However, because the magnitude of covariation between positions varies with the magnitude of positional variation (4), the identification of catalytic residues as part of coevolving networks is problematic for the simple reason that catalytic residues show little sequence variation. Although others have identified putative coevolution between residues involv...

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