Abstract
Assembly of pili in Gram-positive bacteria and their attachment to the cell wall envelope are mediated by sortases. In Bacillus cereus and its close relative Bacillus anthracis, the major pilin protein BcpA is cleaved between the threonine and the glycine of its C-terminal LPXTG motif sorting signal by the pilin-specific sortase D. The resulting acyl enzyme intermediate is relieved by the nucleophilic attack of the side-chain amino group of lysine within the YPKN motif of another BcpA subunit. Cell wall anchoring of assembled BcpA pili requires sortase A, which also cleaves the LPXTG sorting signal of BcpA between its threonine and glycine residues. We show here that sortases A and D require only the C-terminal sorting signal of BcpA for substrate cleavage. Unlike sortase D, which accepts the YPKN motif as a nucleophile, sortase A forms an amide bond between the BcpA C-terminal carboxyl group of threonine and the side-chain amino group of diaminopimelic acid within the cell wall peptidoglycan of bacilli. These results represent the first demonstration of a cell wall anchor structure for pili, which are deposited by sortase A into the envelope of many different microbes.
Highlights
Acyl-enzyme between the active site cysteine residue of sortase and the carboxyl group of threonine [7, 8]
Substrate Properties Control the Fate of Pilin Precursors— Previous work demonstrated that sortase A and sortase D both cleave the major pilin protein precursor BcpA, but left unresolved the substrate requirements of these enzymes
In the presence of sortase D, BcpA is polymerized into pili, whereas sortase A is thought to immobilize pili in the cell wall envelope [4]
Summary
Unlike sortase D, which accepts the YPKN motif as a nucleophile, sortase A forms an amide bond between the BcpA C-terminal carboxyl group of threonine and the sidechain amino group of diaminopimelic acid within the cell wall peptidoglycan of bacilli. Pilin-specific sortases perform a transpeptidation reaction, whereby the C-terminal LPXTG motif sorting signal of the major pilin protein, BcpA in bacilli, is cleaved between the threonine and the glycine residue [4]. Intermediary product of this reaction is a thioester-linked. Conservation of pilin-specific sortase, the YPKN motif, and C-terminal sorting signal in the major subunit of pilin proteins suggests that pilus assembly occurs by a universal mechanism in Gram-positive bacteria [2].
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