Arm and head domain in highly conserved lipoprotein modification enzyme Lgt determine functional diversity among...

The post-translational lipoprotein modification pathway is conserved in bacteria, in which prolipoprotein phosphatidylglycerol diacylglyceryl transferase (Lgt) catalyzes the first and committed step. Due to its essentiality for cell viability in Proteobacteria, its membrane localization, and relative accessibility, Lgt is proposed as a promising target for the development of novel antibiotics. To answer the question of the degree of conservation between Lgt homologs of WHO-listed pathogenic species, we performed evolutionary, structural, and functional analyses. Our data show that Lgt is present in all bacteria and absent from archaea. AlphaFold structural models are similar to the X-ray structure of Lgt from E. coli with most variability and less conserved residues in the arm and head domains between Lgt homologs. Lgt of diderm bacteria, but not of monoderm bacteria, restores growth and viability of an Lgt depletion strain in E. coli. Sequence alignments and site-directed mutagenesis demonstrate that unique conserved residues on arm-2 together with histidine 103 and the periplasmic head domain, determine protein substrate specificity. This large-scale analysis led to the definition of an Lgt motif and an alternative catalytic mechanism. Our results highlight similarities in catalytic mechanism and differences in substrate specificity between Lgt homologs from pathogenic species, with an impact on strategies to develop narrow-spectrum antibiotics targeting Lgt.IMPORTANCEAntimicrobial resistance is a major threat to public health, for which the identification of novel targets and the development of new therapies are urgently needed. The bacterial lipoprotein modification pathway is promising for the exploration of new antibiotics since it is unique to bacteria, essential for bacterial viability and virulence, and accessible to drugs due to the exposed domains of the modification enzymes. In this study, we explored large-scale sequence analysis, structural modeling, and functional assays of the first enzyme in the pathway. Our findings show that the enzyme is highly conserved across distant phyla, that homologous enzymes have similar structures and contain a signature motif composed of invariant essential residues, but that functional conservation divides monoderm and diderm pathogenic bacteria. This correlates with structural variation and differences in substrate specificity, illustrating the potential for the development of narrow-spectrum antibiotics targeting the lipoprotein modification pathway.