Functional Analysis of MmeI from Methanol UtilizerMethylophilus methylotrophus, a Subtype IIC Restriction-Modification Enzyme Related to Type I Enzymes

Abstract

MmeI from6 -adenine ␥-class DNA methyltransferases; (iii) the C-terminal portion (aa 610 to 919) containing a putative target recognition domain. Interestingly, all three domains showed highest similarity to the corresponding elements of type I enzymes rather than to classical type II enzymes. We have found that MmeI variants deficient in restriction activity (D70A, E80A, and K82A) can bind and methylate specific nucleotide sequence. This suggests that domains of MmeI responsible for DNA restriction and modification can act independently. Moreover, we have shown that a single amino acid residue substitution within the putative target recognition domain (S807A) resulted in a MmeI variant with a higher endonucleolytic activity than the wild-type enzyme.Restriction-modification (RM) systems are composed of two enzymatic entities: a restriction endodeoxyribonuclease (REase) that cleaves DNA usually within short, specific sequences and a methyltransferase (MTase) that modifies the same sequence in order to protect the host genomic DNA against the action of the cognate restriction enzyme. Based on their molecular structure, functional features, and cofactors requirements, RM systems can be divided into four distinct types (49). The most complex are enzymes of types I and III (and some of type IV), which function as multisubunit molecular machines that exhibit very complex mechanism of action involving NTP hydrolysis and DNA translocation (reviewed in reference 4). On the other hand, type II RM systems comprise relatively simple independent REase and MTase enzymes whose properties, in particular recognition of short specific nucleotide sequences (4 to 8 bp), have made them indispensable tools of modern molecular biology (45). To date, almost 4,000 type II REases have been identified by screening various Bacteria, Archaea, and viruses and characterizing them biochemically (50).DNA MTases of RM systems belong to several different families within the Rossmann fold methyltransferase superfamily (6). Structural conservation is strong across catalytic domains of all DNA MTases, despite sequence divergence between families and frequent fusions with additional, unrelated domains that are involved in, for example, specific recognition of the target DNA sequence (29). Two major families are the m 5 C MTases, a group of proteins with high mutual sequence similarity and only remote similarity to other MTases (48), and the N-MTases (m 6 A and m 4 C MTases), a large and heterogeneous group of enzymes that exhibit very complex mutual relationships (9,34). The N-MTase family contains not only type II enzymes, but also MTase subunits

Related bifunctional restriction endonuclease-methyltransferase triplets: TspDTI, Tth111II/TthHB27I and TsoI with distinct specificities

“…This domain organization resembles the structure of the recently analysed Type IIC enzymes TspGWI [19] and MmeI [33]), to which TspDTI exhibits only very limited sequence similarity, restricted primarily to the central RFM domain (data not shown). The presence of related domains in a common linear arrangement suggests that all these enzymes evolved from a common ancestor, but that they diverged greatly in all regions except the RFM domain.…”

Structure and operation of the DNA-translocating type I DNA restriction enzymes

“…These classes appear to contain structural domains in common with the type I RM enzymes; namely, endonuclease domains, an HsdM-like subunit, and TRDs, but no motor domains (Dryden 1999;Nakonieczna et al 2009;Shen et al 2011). In these enzymes, the motor domains of HsdR are missing and the endonuclease domain is directly The predicted complete path of the DNA (green dots) through the atomic model of EcoR124I with segments of bound DNA.…”

“…Thus, a type IIB RM enzyme is a ''motor-less'' type I RM system, and a type IIG system is half of a ''motor-less'' type I RM enzyme. Figure 7 compares the relative locations of one endonuclease domain, one HsdM, and the HsdS from the closed form of EcoR124I with the structures of the type IIG enzymes MmeI and BpuSI (Nakonieczna et al 2009;Shen et al 2011). MmeI recognizes the sequence TCCRAC and cuts downstream at N20/N18 or N21/N19.…”

“…The catalytic motifs in the endonuclease domain and HsdM are shown in spacefill. The middle structure shows the structural model of MmeI with bound DNA with the same coloring used for equivalent domains (endonuclease domain, N-terminal domain, MTase catalytic domain, and TRD) (Nakonieczna et al 2009; coordinates from ftp://genesilico.pl/iamb/models/RM.MmeI). The structure on the right shows the crystallographic structure of BpuSI (PDB code: 3s1s) with the same coloring of domains as in the other structures and with an inserted extra domain shown in gray (Shen et al 2011).…”

Crystallization and preliminary crystallographic analysis of the type IIL restriction enzymeMmeI in complex with DNA

“…To better understand the structural basis of DNA recognition and cleavage by type IIL REs, we have crystallized MmeI in complex with DNA. MmeI is a large enzyme (919 amino acids) that encompasses DNA-recognition, methyltransferase and endonuclease activities in the same polypeptide (Boyd et al, 1986;Tucholski et al, 1998;Nakonieczna et al, 2009;Morgan et al, 2008). The enzyme recognizes the asymmetric DNA sequence TCCRAC (where R is a purine) and cleaves the DNA two helical turns away from the recognition sequence: TCCRAC20/18.…”