Abstract
The Sortase family of transpeptidases are found in numerous gram-positive bacteria and involved in divergent physiological processes including anchoring of surface proteins to the cell wall as well as pili assembly. As essential proteins, sortase enzymes have been the focus of considerable interest for the development of novel anti-microbials, however, more recently their function as unique transpeptidases has been exploited for the synthesis of novel bio-conjugates. Yet, for synthetic purposes, SrtA-mediated conjugation suffers from the enzyme’s inherently poor catalytic efficiency. Therefore, to identify SrtA variants with improved catalytic efficiency, we used directed evolution to select a catalytically enhanced SrtA enzyme. An analysis of improved SrtA variants in the context of sequence conservation, NMR and x-ray crystal structures, and kinetic data suggests a novel mechanism for catalysis involving large conformational changes that delivers substrate to the active site pocket. Indeed, using DEER-EPR spectroscopy, we reveal that upon substrate binding, SrtA undergoes a large scissors-like conformational change that simultaneously translates the sort-tag substrate to the active site in addition to repositioning key catalytic residues for esterification. A better understanding of Sortase dynamics will significantly enhance future engineering and drug discovery efforts.
Highlights
The Sortase enzymes are a ubiquitous and highly conserved family of transpeptidases that catalyze the incorporation of cell surface proteins into the prokaryotic cell wall[1,2,3,4]
To identify improved variants of Sortase A (SrtA), which may aid in the further elucidation of the SrtA catalytic mechanism, we utilized the dihydrofolate reductase (DHFR) protein complementation assay originally described by Michnick et al (Fig 1A)[42,43,44]
DHFR function is essential for tetrahydrofolate synthesis and purine biosynthesis in vivo, and the DHFR protein is well conserved from bacteria to humans as demonstrated by the fact that mammalian DHFR can complement DHFR-deficient bacteria
Summary
The Sortase enzymes are a ubiquitous and highly conserved family of transpeptidases that catalyze the incorporation of cell surface proteins into the prokaryotic cell wall[1,2,3,4]. The enzymes can be classified into four sub-families, A-D, based on their sequence conservation and substrate specificity. Each sub-family appears to participate in a unique physiological pathway. While SrtA family members are ubiquitous in gram-positive bacteria and are primarily involved in the coupling of surface proteins required for general physiology, SrtB, SrtC, and SrtD on the other hand, are present in many fewer species and function during the iron deprivation response (SrtB) and pilin formation, respectively[2].
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