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

Abstract

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. Author summaryAntimicrobial resistance is a major threat to public health for which novel targets to develop new therapies is urgently needed. The bacterial lipoprotein modification pathway is promising for exploration of new antibiotics since it is unique to bacteria, it is essential for bacterial viability and virulence, and it is accessible to drugs due to the exposed domains of the modification enzymes. In this study we explored large-scale sequence analysis, structural modelling 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 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.