Structural basis of the membrane intramolecular transacylase reaction responsible for lyso-form lipoprotein synthesis

Abstract

Lipoproteins serve diverse functions in the bacterial cell and some are essential for survival. Some lipoproteins are adjuvants eliciting responses from the innate immune system of the host. The growing list of membrane enzymes responsible for lipoprotein synthesis includes the recently discovered lipoprotein intramolecular transacylase, Lit. Lit creates a lipoprotein that is less immunogenic, possibly enabling the bacteria to gain a foothold in the host by stealth. Here, we report the crystal structure of the Lit enzyme from Bacillus cereus and describe its mechanism of action. Lit consists of four transmembrane helices with an extracellular cap. Conserved residues map to the cap-membrane interface. They include two catalytic histidines that function to effect unimolecular transacylation. The reaction involves acyl transfer from the sn-2 position of the glyceryl moiety to the amino group on the N-terminal cysteine of the substrate via an 8-membered ring intermediate. Transacylation takes place in a confined aromatic residue-rich environment that likely evolved to bring distant moieties on the substrate into proximity and proper orientation for catalysis.

Bacterial Lipoprotein Posttranslational Modifications. New Insights and Opportunities for Antibiotic and Vaccine Development

“…These show convincingly that Lit transfers the ester-linked acyl chain at the sn -2 position of the glyceryl moiety on the substrate directly to the free α-amino group of its N-terminal cysteine ( Figure 10A ). The proposed mechanism, which does not involve the formation of an acyl-enzyme intermediate, is supported by molecular dynamics and quantum mechanics/molecular mechanics simulations ( Olatunji et al, 2021 ). Transfer is of the general acid-base type mechanism involving two highly conserved histidine residues (His85 and His153 in B. cereus , Figure 10B ).…”

“…The crystal structure of Lit from Bacillus cereus was published recently ( Figure 9 ; Olatunji et al, 2021 ). It is a 218 amino acid residue, cone-shaped protein with four TMHs that span the membrane.…”

“…In the crystal structure, two monoolein molecules, originating from the lipid cubic phase used for crystallization, were found within the putative substrate binding pocket. These have been taken as proxies for the two acyl chains in the DA-BLP substrate of Lit ( Olatunji et al, 2021 ).…”

“… Structural features of Lit from B. cereus. The structure of Lit (PDP code 7B0P, Olatunji et al, 2021 ) is shown in two orientations viewed from the membrane plane. The protein is colored to highlight the pseudo-structural symmetry between the N-terminal membrane domain 1 (MD1) and extramembrane globular domain 1 (EGD1) (shades of blue) and the C-terminal MD2 and EGD2 (shades of red).…”

“…The mechanism of the intramolecular acyl transfer reaction catalyzed by Lit was investigated by mass spectrometry and nuclear magnetic resonance spectroscopy using deuterium-labeled lipopeptide and BLP substrates ( Armbruster et al, 2020 ; Olatunji et al, 2021 ). These show convincingly that Lit transfers the ester-linked acyl chain at the sn -2 position of the glyceryl moiety on the substrate directly to the free α-amino group of its N-terminal cysteine ( Figure 10A ).…”