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

Lipoproteins are important components of the bacterial cell envelope that itself is an excellent target for antibiotics. The post-translational lipoprotein modification pathway is conserved in bacteria in which prolipoprotein phosphatidylglycerol diacylglyceryl transferase (Lgt) catalyses the first and committed step. Due to its essentiality for cell viability in proteobacteria its membrane localisation and relative accessibility, Lgt is proposed as promising target for the development of novel antibiotics. To answer the question of the degree of conservation between Lgt homologues 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. Alpha Fold 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. Lgt of proteobacteria but not of firmicutes restore growth and viability of a Lgt depletion strain in E. coli . Sequence alignments and site-directed mutagenesis demonstrate that unique conserved residues on arm-2 together with histidine 103 determine protein substrate specificity. This large-scale analysis led to the definition of a 13-residue Lgt motif and an alternative catalytic mechanism. Our results highlight similarities in catalytic mechanism and differences in substrate specificity between Lgt homologues from pathogenic species with impact on strategies to develop narrow-spectrum antibiotics targeting Lgt.