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

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.

Genetic heterogeneity and respiratory chain enzyme analysis in pediatric Indian patients with mitochondrial disorder: Report of novel variants in POLG1 gene and their functional implication using molecular dynamic simulation.

Mitochondrial disorders are a heterogeneous group of disorders caused by mutations in the mitochondrial DNA or in nuclear genes encoding the mitochondrial proteins and subunits. Polymerase Gamma (POLG) is a nuclear gene and mutation in the POLG gene are one of the major causes of inherited mitochondrial disorders. In this study, 15 pediatric patients, with a wide spectrum of clinical phenotypes were screened using blood samples (n = 15) and muscle samples (n = 4). Respiratory chain enzyme analysis in the muscle samples revealed multi-complex deficiencies with Complex I deficiency present in (1/4) patients, Complex II (2/4), Complex III (3/4) and Complex IV (2/4) patients. Multiple large deletions were observed in 4/15 patients using LR-PCR. Whole exome sequencing (WES) revealed a compound heterozygous mutation consisting of a POLG1 novel variant (NP_002684.1:p.Trp261X) and a missense variant (NP_002684.1:p. Leu304Arg) in one patient and another patient harboring a novel homozygous POLG1 variant (NP_002684.1:p. Phe750Val). These variants (NP_002684.1:p. Leu304Arg) and (NP_002684.1:p. Phe750Val) and their interactions with DNA were modelled using molecular docking and molecular dynamics (MD) simulation studies. The protein conformation was analyzed as root mean square deviation (RMSD), root mean square fluctuation (RMSF) which showed local fluctuations in the mutants compared to the wildtype. However, Solvent Accessible Surface Area (SASA) significantly increased for NP_002684.1:p.Leu304Arg and decreased in NP_002684.1:p.Phe750Val mutants. Further, Contact Order analysis indicated that the Aromatic-sulfur interactions were destabilizing in the mutants. Overall, these in-silico analysis has revealed a destabilizing mutations suggesting pathogenic variants in POLG1 gene.