Bacterial lipoproteins constitute 2-5% of the bacterial proteome and are important for many aspects of bacterial cell envelope physiology. Lipoproteins are translated as precursor lipoproteins (preproLpp) in the cytosol and directed to the inner membrane by signal peptide. In the membrane, preproLpp undergo a unique lipidation process mediated by three sequential enzymes: (i) lipoprotein diacylglyceryl transferase (Lgt), (ii) lipoprotein signal peptidase II (LspA), and (iii) lipoprotein Nacyltransferase (Lnt). The process begins with Lgt transferring a diacylglyceryl moiety to the conserved cysteine residue within the lipobox motif (LVI/ASTVI/GAS/C) of preproLpp. As Lgt is often essential for bacterial viability and absent in humans, it represents a promising target for novel antimicrobial development. To explore this therapeutic potential, we performed multiple sequence alignment (MSA) of Lgt. This revealed a critical distinction between proteobacterial and Grampositive bacterial Lgt enzymes. While proteobacteria utilise (Histidine/His 103) H103 as a catalytic base for cysteine deprotonation, Gram-positive bacteria employ aromatic residues (Tyrosine in GBS, Tryptophan in Mycobacterium tuberculosis, Phenylalanine in others) at this position. Based on X-ray crystal structure of E.coli Lgt published by Mao and colleagues, we sought to understand the molecular basis of Lgt function through computational investigation of its catalytic mechanism. X-ray crystal structure available provided crucial structural insights into Lgt, but notably lacked the preproLpp substrate, limiting mechanistic understanding. To address this gap, we employed molecular docking, molecular dynamics (MD) simulations, and QM/MM calculations to generate the first ever catalytically productive complex of Lgt with preproLpp. QM/MM calculations demonstrated that H103 functions as a catalytic base, abstracting a proton from the conserved cysteine thiol of preproLpp to generate a nucleophilic thiolate. This activated cysteine then attacks the C3 carbon of phosphatidylglycerol via sn2 mechanism, resulting in concerted diacylglyceryl transfer and release of glycerol-1-phosphate. This represents the first atomistic description of the complete Lgt catalytic cycle with both substrates present, establishing the molecular basis for lipoprotein lipidation. The computational analysis also revealed that (Arginine, R) R143 and R239 stabilise the glycerol-1- phosphate head group of PG substrate. Furthermore, the L6–7 loop, regulated by R236, functions as a gatekeeper facilitating product release and substrate exchange. The computational approach was then extended to examining catalytic variations across different bacterial phyla. MD simulations of AlphaFold modelled GBS and M. tuberculosis Lgt structures demonstrated stable enzymesubstrate complexes with the catalytic cysteine positioned within 3.5A and 4.5A of the PG C3 carbon, respectively. Notably, these Gram-positive variants achieve cysteine activation through electrostatic stabilisation by conserved arginine residues (R143, R239, R246) rather than conventional general base catalysis, enabling thiolate formation necessary for diacylglyceryl transfer. In addition to the computational studies, we also conducted experimental study to characterise the phenotypes of Lgt mutant of Streptococcus agalactiae (GBS) A909. These studies demonstrated that lgt deletion did not significantly impair growth under standard conditions but reduced survival time during stationary and death phases, suggesting Lgt's role in stress tolerance. The Δlgt mutant of GBS exhibited significantly decreased cell surface hydrophobicity, indicating compromised cell envelope integrity due to the lack of lipidation. Importantly, the mutant showed increased susceptibility to aminoglycoside antibiotics (gentamicin, streptomycin, kanamycin) with ≥2 fold reduction in MIC values. While glucose supplementation enhanced biofilm formation in both wild-type and mutant strains, no significant difference was observed between them, and D-amino acids showed no antibiofilm activity. To conclude, the integration of experimental validation, bioinformatics, computational modelling confirms that Lgt mediated lipidation is significant across all bacterial phyla, despite mechanistic variations between Gram-positive and Gram-negative bacteria. The identification of distinct catalytic strategies direct base catalysis in proteobacteria versus electrostatic stabilisation in Gram-positive bacteria provides crucial insights for structure-based drug design. Together, these mechanistic insights establish a foundation for developing broad spectrum or phyla specific antimicrobial agents targeting this critical enzyme in bacterial lipoprotein biosynthesis.
- Bacterial cell envelope proteins
- Signal peptide processing and post translational modification of proteins
- Enzyme catalytic mechanism and novel antimicrobials
- Lipid modification of bacterial proteins and their roles in bacteria
Investigations of lipoprotein biosynthesis in bacteria: structural-functional relationship of prolipoprotein diacyglyceryl transferase
Munir, M. (Author). 19 Mar 2026
Student thesis: Doctoral Thesis