Functional Identification Of Metal-β-Lactamase And Its Interaction With Chaperone Protein In Mycobacterium Tuberculosis

Mycobacterium tuberculosis is a highly infectious pathogen that can cause TB in humans and animals. M. tuberculosis has a very strong resistance as they can synthesize some hydrolytic enzyme (egβ-lactamase), or a mutation in drug targets. In this study, we found that the M.tuberculosis Rv2752c possessed both lactamase activity and RNase activity. The results are as follows:(1) The bioinformatics analysis shows that Rv2752c belongs to metal-β-lactamase family ofβ-CASP subfamily and also contains RMMBL domain, indicating that Rv2752c may has double function--lactamase and RNase. A high homology between Rv2752c and RNaseJ was demonstrated through an amino acid sequence alignment assay. (2) We made several site-specific gene mutations and a series of deletion mutations, and further expressed and purified their proteins. We confirmed that Rv2752c had both lactamase and RNase activities. Two residues, D184 and H397, were found to be essential for binding of metal ions and for the dual activities of the protein. The deletion of 100 amino acid residues in C-terminus also resulted in the lack of both lactamases and RNase activity of Rv2752c. (3) Using bacterial two-hybrid screening and PULL-DOWN, we confirmed Rv2752c interacted with Rv2373c, and further found that Rv2373c inhibited the double activities of Rv2752c.This is the first time report on a mycobacterial protein with both lactamase and RNase. This finding improves our understanding in the mature mRNA processing and drug resistance in M.tuberculosis, and provides many information on the the relationship between structure and function of the microbial lactamase.