RNA-hydrolyzing activity of metallo-β-lactamase IMP-1.

Yoshiki Kato,Mineaki Seki,Akiko Shimizu-Ibuka,Masayuki Takahashi,Masayuki Nashimoto,Israel Silman

doi:10.1371/journal.pone.0241557

Yoshiki Kato, Mineaki Seki + Show 4 more

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https://doi.org/10.1371/journal.pone.0241557

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Abstract

Metallo-β-lactamases (MBLs) hydrolyze a wide range of β-lactam antibiotics. While all MBLs share a common αβ/βα-fold, there are many other proteins with the same folding pattern that exhibit different enzymatic activities. These enzymes, together with MBLs, form the MBL superfamily. Thermotoga maritima tRNase Z, a tRNA 3′ processing endoribonuclease of MBL-superfamily, and IMP-1, a clinically isolated MBL, showed a striking similarity in tertiary structure, despite low sequence homology. IMP-1 hydrolyzed both total cellular RNA and synthetic small unstructured RNAs. IMP-1 also hydrolyzed pre-tRNA, but its cleavage site was different from those of T. maritima tRNase Z and human tRNase Z long form, indicating a key difference in substrate recognition. Single-turnover kinetic assays suggested that substrate-binding affinity of T. maritima tRNase Z is much higher than that of IMP-1.

Highlights

Antibiotic-resistant bacteria, Gram-negative strains, have developed a major defense mechanism against β-lactam-based antibiotics by producing β-lactamases (EC 3.5.2.6) that hydrolyze β-lactam rings
This difference in activity profiles may be associated with Zn2+ coordination differences at their catalytic sites as B1 and B3 enzymes exhibit maximum activity when two Zn2+ ions are bound to their active sites, but B2 enzymes are inhibited by the binding of more than one Zn2+ ion [1]
We investigated the hydrolytic activity of IMP-1 against two 50-6-carboxyfluorescein-labeled unstructured RNAs (usRNAs): 24-nt usRNA1 and 22-nt usRNA9

Summary

Introduction

Antibiotic-resistant bacteria, Gram-negative strains, have developed a major defense mechanism against β-lactam-based antibiotics by producing β-lactamases (EC 3.5.2.6) that hydrolyze β-lactam rings. TmRZ and some other tRNase Z enzymes can cleave unstructured RNAs (usRNAs) in addition to pre-tRNA. To determine kchem (rate constant) and Kd (dissociation constant) for the RNase activity of IMP-1 and TmRZ, single-turnover assays were performed, in which kobs (apparent rate constant) was measured at different enzyme concentrations [23].

Results

Conclusion