Conserved Amino Acid Moieties of Candidatus Desulforudis audaxviator MazF Determine Ribonuclease Activity and Specificity

Abstract

The toxin-antitoxin (TA) system, inherent to various prokaryotes, plays a critical role in survival and adaptation to diverse environmental stresses. The toxin MazF, belonging to the type II TA system, functions as a sequence-specific ribonuclease that recognizes 3 to 7 bases. In recent studies, crystallographic analysis of MazFs from several species have suggested the presence of amino acid sites important for MazF substrate RNA binding and for its catalytic activity. Herein, we characterized MazF obtained from Candidatus Desulforudis audaxviator (MazF-Da) and identified the amino acid residues necessary for its catalytic function. MazF-Da, expressed using a cell-free protein synthesis system, is a six-base-recognition-specific ribonuclease that preferentially cleaves UACAAA sequences and weakly cleaves UACGAA and UACUAA sequences. We found that MazF-Da exhibited the highest activity at around 60°C. Analysis using mutants with a single mutation at an amino acid residue site that is well conserved across various MazF toxins showed that G18, E20, R25, and P26 were important for the ribonuclease activity of MazF-Da. The recognition sequence of the N36A mutant differed from that of the wild type. This mutant cleaved UACAAG sequences in addition to UACAAA sequences, but did not cleave UACGAA or UACUAA sequences, suggesting that Asn36 affects the loosening and narrowing of MazF-Da cleavage sequence recognition. Our study posits UACAAA as the recognition sequence of MazF-Da and provides insight into the amino acid sites that are key to its unique enzymatic properties.

Helicobacter macacaeMazF interplays with Escherichia coli homologs and enhances antibiotic tolerance

“…The isoelectric point of MazEhm and MazFhm are 9.06 and 9.38, respectively, both higher than pH 7.0, which is different from classical MazEF systems, whose toxin MazF is usually acidic, and the cognate antitoxin are basic at neutral pH 21,22 . MazFhm shared 42.5% amino acid identity with MazFec, and MazEhm shared 30.6% amino acid identity with MazEec, which is comparable to similarities of MazF homologs from other species with MazFec 21,22,30 …”

“…21,22 MazFhm shared 42.5% amino acid identity with MazFec, and MazEhm shared 30.6% amino acid identity with MazEec, which is comparable to similarities of MazF homologs from other species with MazFec. 21,22,30…”

MazEF Homologs in Symbiobacterium thermophilum Exhibit Cross-Neutralization with Non-Cognate MazEFs

“…Considering the above homology, the cross-neutralization between MazF and non-cognate MazE was examined, revealing the obvious suppression of MazE-bs on MazF-sth. The structure mechanism was analyzed by multiple sequence alignment (MSA) [ 11 , 13 , 14 , 16 , 17 , 18 , 19 , 20 , 25 , 26 , 41 , 42 , 43 , 44 , 45 , 46 , 47 ] ( Figure S5 ) combined with the simulation of the MazEF-sth complex structure via AlphaFold2 [ 48 ] ( Figure S6 ). Focus was placed on the interface; next, regions of the MazF-sth dimer interacting with MazE-sth were estimated as follows and highlighted with red/blue squares on MSA ( Figures S5 and S6 ).…”

“…The specific recognition has also been proposed to be decided by several amino acid residues. Arg25 and Thr48 of MazF from Bacillus subtilis (MazF-bs) are essential, especially for enzymatic activity [ 15 ]; moreover, the mutation into Asn36 of MazF from Candidatus Desulforudis audaxviator shifted the recognition sequence [ 16 ]. Yet notably, as represented by MazF-bs, several MazFs from Gram-positive bacteria have been discovered successively to recognize and cleave at the exact same UACAU motif [ 17 , 18 , 19 , 20 ].…”

Arg-73 of the RNA endonuclease MazF in Salmonella enterica subsp. arizonae contributes to guanine and uracil recognition in the cleavage sequence

“…Further, the guanidinium group of Arg and the hydroxyl group of Thr act nucleophilically on the phosphate bond of RNA, causing transphosphorylation and forming a 2′3′-cyclic phosphate and 5′-hydroxyl group ( 14 , 15 ). Arg is an essential residue for RNA cleavage activity; hence, when Arg-25 of Candidatus Desulforudis audaxviator MazF (MazF-da) was replaced with Ala, there was a complete loss in cleavage activity ( 16 ). Structural analysis also revealed an interaction between MazF residues and RNA bases recognized by MazF.…”