Structure of Type IIL Restriction-Modification Enzyme MmeI in Complex with DNA Has Implications for Engineering New Specificities

Abstract

The creation of restriction enzymes with programmable DNA-binding and -cleavage specificities has long been a goal of modern biology. The recently discovered Type IIL MmeI family of restriction-and-modification (RM) enzymes that possess a shared target recognition domain provides a framework for engineering such new specificities. However, a lack of structural information on Type IIL enzymes has limited the repertoire that can be rationally engineered. We report here a crystal structure of MmeI in complex with its DNA substrate and an S-adenosylmethionine analog (Sinefungin). The structure uncovers for the first time the interactions that underlie MmeI-DNA recognition and methylation (5’-TCCRAC-3’; R = purine) and provides a molecular basis for changing specificity at four of the six base pairs of the recognition sequence (5’-TCCRAC-3’). Surprisingly, the enzyme is resilient to specificity changes at the first position of the recognition sequence (5’-TCCRAC-3’). Collectively, the structure provides a basis for engineering further derivatives of MmeI and delineates which base pairs of the recognition sequence are more amenable to alterations than others.

Isolation of five type IIG restriction modification (RM) enzyme genes with different DNA recognition sites from a single environmental DNA sample

“…The next step should be the characterization of more Mae843ORF8180-like genes in other local cyanobacteria rich samples. On this basis, predictions of the interactions between the amino-acid and recognized DNA bases could be made in order to be able to engineer these enzymes and generate the desired recognition sequences Callahan et al, 2016). Analysing genes from bulk natural DNA could be more efficient than from in vitro grown cells.…”

Detect Positive Selection in Type IIL Restriction Enzyme Using PAML

“…The molecular basis of sequence recognition in type IIL RE has been elucidated. Amino acids that correlated with the specificity changes were identified and 7 of them (corresponding to positions 645,751,773,774,806,808,810 in MmeI, Figure 1) were confirmed to cause specificity change at base 2, 3, 4 and 6 by mutagenesis experiment and the structure of enzyme-DNA complex (Morgan & Luyten, 2009;Callahan et al, 2016). Because those specificity changes correspond to functional changes in nature, the 7 sites are expected to be under positive selection to match the rapid evolution of the restriction sites in phages.…”

“…Most of mutants at these sites retained comparable activities (Morgan & Luyten, 2009;Callahan et al, 2016), suggesting few if any sites other than these 7 amino acids contributed to sequence recognition. The 7 sites were all located within the TRD and close to DNA ( Figure 1B).…”

“…For this reason, there is strong constraint for evolutionary changes for type II RM, since synchronous changes are rare. However, in one family of type II RM proteins (type IIL REs), the two functional modules are merged into one protein and they share one target DNA recognition domain (TRD) (Callahan et al, 2016). As a result, the TRD of type IIL REs should be more tolerant to specificity changes and amenable to positive selection.…”

“…None of them were located close to DNA in the structure or correlated significantly with specificity changes (Morgan & Luyten, 2009;Callahan et al, 2016). Because amino acids not making direct contact with DNA can also influence specificity (Lukacs et al, 2000), I…”