Substrate Recognition and Activity Regulation of the Escherichia coli mRNA Endonuclease MazF

Abstract

Escherichia coli MazF (EcMazF) is the archetype of a large family of ribonucleases involved in bacterial stress response. The crystal structure of EcMazF in complex with a 7-nucleotide substrate mimic explains the relaxed substrate specificity of the E. coli enzyme relative to its Bacillus subtilis counterpart and provides a framework for rationalizing specificity in this enzyme family. In contrast to a conserved mode of substrate recognition and a conserved active site, regulation of enzymatic activity by the antitoxin EcMazE diverges from its B. subtilis homolog. Central in this regulation is an EcMazE-induced double conformational change as follows: a rearrangement of a crucial active site loop and a relative rotation of the two monomers in the EcMazF dimer. Both are induced by the C-terminal residues Asp-78 -Trp-82 of EcMazE, which are also responsible for strong negative cooperativity in EcMazE-EcMazF binding. This situation shows unexpected parallels to the regulation of the F-plasmid CcdB activity by CcdA and further supports a common ancestor despite the different activities of the MazF and CcdB toxins. In addition, we pinpoint the origin of the lack of activity of the E24A point mutant of EcMazF in its inability to support the substrate binding-competent conformation of EcMazF.

Characterization of a Deinococcus radiodurans MazF: A UACA‐specific RNA endoribonuclease

“…To the best of our knowledge, this is the first MazF that specifically recognizes the U^ACA sequence (Masuda & Inouye, ; Schifano & Woychik, ). Recently, Zorzini et al., () determined the crystal structure of E. coli MazF (MazFec) in complex with the substrate analogue d(A 1 U 2 A 3 C 4 A 5 U 6 A 7 ), and reinforced the notion that MazFec recognizes ACA triplet strictly and that MazFec cleaves the substrate at the position of ^ACA and A^CA (Miyamoto et al., ; Vesper et al., ; Zhang, Zhang, Hara, Kato, & Inouye, a; Zhang et al., ). They mentioned that the MazFec recognition site could be divided into two different regions: first, the one where dU 2 is located, which is called the upstream binding site; and second, the one that accommodates d(A 3 C 4 A 5 U 6 ), which is called the downstream binding groove.…”

Structure, Biology, and Therapeutic Application of Toxin–Antitoxin Systems in Pathogenic Bacteria

“…The MazF structures in complex with uncleavable substrate analogues from B. subtilis and E. coli are shown in Figure 2B,C. The structural comparison reveals that they have a common mode of substrate recognition on a large interaction surface [167]. However, the substrate binding of E. coli MazF is achieved by fewer hydrogen bonds to specific RNA bases and poorer surface complementarity than that of B. subtilis MazF, possibly explaining the relaxed substrate specificity of E. coli MazF.…”

Biochemical characterization of mt‐PemIK, a novel toxin‐antitoxin system in Mycobacterium tuberculosis

“…Sequence alignments shown some relatively conserved amino acids among in the putative active centers. For instance, H19 is conserved in the PemK family, and Q21 and R25 in mt‐PemK are corresponding to Q25 and R29 in MazF, which are conserved residues in MazF family and involved in MazF RNA cleavage activity . The mt‐PemK mutant with substitution mutation on anyone of these three residues (H19A, Q21A, or R25A) lost its toxic ability (Fig.…”