Structure and reconstitution of a hydrolase complex that may release peptidoglycan from the membrane after polymerization

Abstract

Bacteria are surrounded by a peptidoglycan cell wall that is essential for their survival 1 . During cell wall assembly, a lipid-linked disaccharide-peptide precursor called Lipid II is polymerized and crosslinked to produce mature peptidoglycan. As Lipid II is polymerized, nascent polymers remain membrane-anchored at one end and the other end becomes crosslinked to the matrix 2 – 4 . A longstanding question is how bacteria release newly synthesized peptidoglycan strands from the membrane to complete the synthesis of mature peptidoglycan. Here we show that a Staphylococcus aureus cell wall hydrolase and a membrane protein containing eight transmembrane helices form a complex that may function as a peptidoglycan release factor. The complex cleaves nascent peptidoglycan internally to produce free oligomers as well as lipid-linked oligomers that can undergo further elongation. The polytopic membrane protein, which is similar to a eukaryotic CAAX protease, controls the length of these products. A 2.6 Å resolution structure of the complex shows that the membrane protein scaffolds the hydrolase to orient its active site for cleavage of the glycan strand. We propose that this complex serves to detach newly-synthesized peptidoglycan polymer from the cell membrane to complete integration into the cell wall matrix.

Biochemical reconstitution defines new functions for membrane-bound glycosidases in assembly of the bacterial cell wall

“…This allows full integration of new strands into the mature cell wall while leaving a competent lipid-linked substrate in the membrane that can be extended once it rebinds to a peptidoglycan synthase. 9,38,39 We favor the hypothesis that MpgA and MpgB, like SagB, serve as peptidoglycan release factors ( Figure 6). These proteins are anchored to the membrane and cleave nascent peptidoglycan bonds that are found at a distance from the lipid anchor that is compatible with the estimated distance of their active sites from the membrane.…”

“…In E.coli and S. aureus, ß-lactam hyper-sensitivity in deletion mutants has been used to identify cleavage enzymes that act early in the cell wall assembly pathway. 9,[19][20][21] Hypersensitivity is notable because deleting cell wall hydrolases often makes cells more rather than less tolerant of ß-lactam treatment, evidently because reducing cell wall degrading activity is advantageous (which was not certified by peer review) is the author/funder. All rights reserved.…”

“…In S. aureus, the membrane anchored N-acetylglucosaminidase SagB has been proposed to perform this role by forming a complex with a partner protein that controls cleavage site selection on the nascent peptidoglycan strand. 9 Here, we have investigated two putative cell wall cleaving enzymes in Streptococcus pneumoniae that were previously reported to play roles in cell wall integrity. S. pneumoniae is an opportunistic pathogen that colonizes mucosa in the upper respiratory tract and can lead to a wide (which was not certified by peer review) is the author/funder.…”

“…In S. aureus , the membrane anchored N-acetylglucosaminidase SagB has been proposed to perform this role by forming a complex with a partner protein that controls cleavage site selection on the nascent peptidoglycan strand. 9…”

“…MpgA and MpgB cleave nascent peptidoglycan at different distances from the lipid anchor to permit full integration of the released polymer into the cell wall. The lipid-linked products are substrates for further extension bypeptidoglycan synthases 9,38,39.…”