The Sortase A Enzyme That Attaches Proteins to the Cell Wall of Bacillus anthracis Contains an Unusual Active Site Architecture

Abstract

The pathogen Bacillus anthracis uses the Sortase A (SrtA) enzyme to anchor proteins to its cell wall envelope during vegetative growth. To gain insight into the mechanism of protein attachment to the cell wall in B. anthracis we investigated the structure, backbone dynamics, and function of SrtA. The NMR structure of SrtA has been determined with a backbone coordinate precision of 0.40 ؎ 0.07 Å . SrtA possesses several novel features not previously observed in sortase enzymes including the presence of a structurally ordered amino terminus positioned within the active site and in contact with catalytically essential histidine residue (His 126 ). We propose that this appendage, in combination with a unique flexible active site loop, mediates the recognition of lipid II, the second substrate to which proteins are attached during the anchoring reaction. pK a measurements indicate that His 126 is uncharged at physiological pH compatible with the enzyme operating through a "reverse protonation" mechanism. Interestingly, NMR relaxation measurements and the results of a model building study suggest that SrtA recognizes the LPXTG sorting signal through a lock-in-key mechanism in contrast to the prototypical SrtA enzyme from Staphylococcus aureus.

Antiinfective therapy with a small molecule inhibitor of Staphylococcus aureus sortase

“…The LPXTG motif is conserved in a wide variety of surface proteins (35); however, the chemical structure of the peptidoglycan crossbridge varies between different bacterial species (36). Although structurally similar (37)(38)(39)(40), the amino acid identity between sortases from different Gram-positive bacteria is limited to a few key residues at or near the active site (34). We therefore asked whether compound 6e can inhibit sortase from three different microbes: Bacillus anthracis, Streptococcus pneumoniae, and Streptococcus pyogenes.…”

Structure of the Bacillus anthracis Sortase A Enzyme Bound to Its Sorting Signal

“…Mutations were made using the QuikChange method (Agilent, San Diego, CA) and confirmed by DNA sequencing. Mutants, wild-type Ba SrtA (amino acid residues Asp 57 -Lys 210 ), and wild-type Ba SrtA ⌬64 (amino acid residues Asp 65 -Lys 210 ) were purified as described previously (23). In vitro substrate hydrolysis assay was performed as previously described (57).…”

“…However, it is not well understood how sortases recognize their sorting signal and nucleophile substrates, because nearly all sortase structures reported to date have been determined in the apo-state. Previously we discovered that the class A Ba SrtA sortase contains a unique N-terminal appendage that wraps around the body of the protein to contact the active site of the enzyme (23). However, the function of this appendage in catalysis remained unknown.…”

Impact of Lactobacillus plantarum Sortase on Target Protein Sorting, Gastrointestinal Persistence, and Host Immune Response Modulation

“…Sortases typically harbor a conserved histidine and a TLXTC motif in their active sites, in which replacement of the single conserved cysteine with alanine has been shown to abolish enzyme activity in Staphylococcus aureus (2). Based on primary sequences and substrate specificities, sortases are grouped into four classes (A to D), of which sortase A (SrtA) is referred to as the housekeeping sortase (3,4). The role of SrtA in surface protein sorting was initially described in S. aureus (5,6), where it was found to recognize proteins bearing the conserved C-terminal cell wall sorting signal LPXTG (7).…”