Structure predictions and functional insights into Amidase_3 domain containing N-acetylmuramyl-L-alanine amidases from Deinococcus indicus DR1

Abstract

Background: N-acetylmuramyl-L-alanine amidases are cell wall modifying enzymes that cleave the amide bond between the sugar residues and stem peptide in peptidoglycan. Amidases play a vital role in septal cell wall cleavage and help separate daughter cells during cell division. Most amidases are zinc metalloenzymes, and E. coli cells lacking amidases grow as chains with daughter cells attached to each other. In this study, we have characterized two amidase enzymes from Deinococcus indicus DR1. D. indicus DR1 is known for its high arsenic tolerance and unique cell envelope. However, details of their cell wall biogenesis remain largely unexplored. Results: We have characterized two amidases Ami1Di and Ami2Di from D. indicus DR1. Both Ami1Di and Ami2Di suppress cell separation defects in E. coli amidase mutants, suggesting that these enzymes are able to cleave septal cell wall. Ami1Di and Ami2Di proteins possess the Amidase_3 catalytic domain with conserved –GHGG- motif and Zn2+ binding sites. Zn2+- binding in Ami1Di is crucial for amidase activity. AlphaFold2 structures of both Ami1Di and Ami2Di were predicted, and Ami1Di was a closer homolog to AmiA of E. coli. Conclusion: Our results indicate that Ami1Di and Ami2Di enzymes can cleave peptidoglycan, and structural prediction studies revealed insights into the activity and regulation of these enzymes in D. indicus DR1.