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Most eukaryotic proteins are N-terminally acetylated by a set of Nα acetyltransferases (NATs). This ancient and ubiquitous modification plays a fundamental role in protein homeostasis, while mutations are linked to human diseases and phenotypic defects. In particular, Naa50 features species-specific differences, as it is inactive in yeast but active in higher eukaryotes. Together with NatA, it engages in NatE complex formation for cotranslational acetylation. Here, we report Naa50 homologs from the filamentous fungi Chaetomium thermophilum and Neurospora crassa with significant N- and C-terminal extensions to the conserved GNAT domain. Structural and biochemical analyses show that CtNaa50 shares the GNAT structure and substrate specificity with other homologs. However, in contrast to previously analyzed Naa50 proteins, it does not form NatE. The elongated N-terminus increases Naa50 thermostability and binds to dynein light chain protein 1, while our data suggest that conserved positive patches in the C-terminus allow for ribosome binding independent of NatA. Our study provides new insights into the many facets of Naa50 and highlights the diversification of NATs during evolution. [ABSTRACT FROM AUTHOR]

Paromomycin, an aminoglycoside antibiotic, is an effective treatment for VL (visceral leishmaniasis) in India. The modification of aminoglycoside antibiotics by enzymes such as aminoglycoside acetyltransferases is the predominant mechanism of resistance to antibiotics in bacterial system. In the present study, we identified and characterized LdATLP (an acetyltransferase-like protein) and elucidated its role in paromomycin resistance in Leishmania donovani . Gene encoding LdATLP was consistently up-regulated (>2fold) in three distinct paromomycin resistant in comparison with sensitive parasites, although the gene sequence was identical in the two. In silico analysis revealed that LdATLP consisted of conserved GNAT (GCN5-related N-Acetyltransferase) domain which is characteristic of aminoglycoside N-acetyltransferases. Evolutionary relationship among LdATLP of Leishmania and aminoglycoside acetyltransferases of bacteria was established by phylogenetic analysis. The 3D structure of LdATLP , predicted by ab-initio modeling, constituted 6 α-helices and 6 β-sheets. A few residues, such as R175, R177, E196, R197, V198, V200, K202, R205, C206, D208, G210, R211, R215, A234, S237, S238, K239, D240, F241 and Y242 of GNAT domain were predicted to be present at active site. Molecular docking of LdATLP with paromomycin or indolicidin (broad spectrum inhibitor of aminoglycoside modifying enzymes), followed by molecular dynamics simulation of docked complex suggested that both paromomycin and indolicidin bind to LdATLP with comparable free energy of binding . In vitro studies revealed that in the presence of indolicidin, paromomycin resistant parasites exhibited reversion of phenotype into sensitive parasites with marked increase in paromomycin susceptibility, suggesting the role of LdATLP in paromomycin resistance.Communicated by Ramaswamy H. Sarma.