Staphylococcus aureus Sortase Transpeptidase SrtA:  Insight into the Kinetic Mechanism and Evidence for a Reverse Protonation Catalytic Mechanism

Abstract

The Staphylococcus aureus transpeptidase SrtA catalyzes the covalent attachment of LPXTG-containing virulence and colonization-associated proteins to cell-wall peptidoglycan in Gram-positive bacteria. Recent structural characterizations of staphylococcal SrtA, and related transpeptidases SrtB from S. aureus and Bacillus anthracis, provide many details regarding the active site environment, yet raise questions with regard to the nature of catalysis and active site cysteine thiol activation. Here we re-evaluate the kinetic mechanism of SrtA and shed light on aspects of its catalytic mechanism. Using steady-state, pre-steady-state, bisubstrate kinetic studies, and high-resolution electrospray mass spectrometry, revised steady-state kinetic parameters and a ping-pong hydrolytic shunt kinetic mechanism were determined for recombinant SrtA. The pH dependencies of kinetic parameters k(cat)/K(m) and k(cat) for the substrate Abz-LPETG-Dap(Dnp)-NH(2) were bell-shaped with pK(a) values of 6.3 +/- 0.2 and 9.4 +/- 0.2 for k(cat) and 6.2 +/- 0.2 and 9.4 +/- 0.2 for k(cat)/K(m). Solvent isotope effect (SIE) measurements revealed inverse behavior, with a (D)2(O)k(cat) of 0.89 +/- 0.01 and a (D)2(O)(k(cat)/K(m)) of 0.57 +/- 0.03 reflecting an equilibrium SIE. In addition, SIE measurements strongly implicated Cys184 participation in the isotope-sensitive rate-determining chemical step when considered in conjunction with an inverse linear proton inventory for k(cat). Last, the pH dependence of SrtA inactivation by iodoacetamide revealed a single ionization for inactivation. These studies collectively provide compelling evidence for a reverse protonation mechanism where a small fraction (ca. 0.06%) of SrtA is competent for catalysis at physiological pH, yet is highly active with an estimated k(cat)/K(m) of >10(5) M(-)(1) s(-)(1).

Engineering the Substrate Specificity of Staphylococcus aureus Sortase A

“…Slightly higher substrate concentrations (2.7 mM) were used than would ideally be the case; however, given the low levels of activity of several of the enzymes being measured, it was necessary to use low millimolar concentrations of substrate in order to obtain detectable levels of product using our HPLC-based assay. (33,39). By contrast, SrtB had no detectable activity against the LPETG substrate, even after 12 h at 37°C and an enzyme concentration of 94.8 M. This is consistent with earlier studies in which no LPETG-cleaving activity was observed for SrtB in vitro (22).…”

“…3). For comparison, previous studies have reported a k cat of 0.28 Ϯ 0.02 s Ϫ1 for SrtA ⌬N24 and a K m of 7.3 Ϯ 1 mM for SrtA ⌬N24 with Abz-LPETG-Dap(DNP)-NH 2 (39). These values show that SrtB has a K m for this substrate that is within the error of that measured for SrtA but a k cat that is ϳ500-fold lower.…”

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

“…15 Nfiltered (F2) 13 C-edited NOESY-HSQC and (F1) 13 C, 15 N-filtered (F2) 15 N-edited NOESY-HSQC (30), and two-dimensional (F1) 13 C-filtered NOESY spectra (29). The majority of and dihedral angle restraints were obtained using the program TALOS (31).…”

“…Additional angle restraints were obtained by measuring 3 J HN␣ values from HNHA spectrum (32), and additional angle restraints were obtained by analyzing threedimensional 15 N-edited NOESY spectrum (33). 1 angles for dihedral angle restraints and stereotypical chemical shift assignments of ␤-methylene protons were obtained by analyzing 15 N-TOCSY-HSQC, HNHB, HN(CO)HB, and 15 N-ROESY spectra (34,35).…”

“…The C-terminal charged tail presumably retards export, positioning the protein for processing by the extracellular membrane-associated Sa SrtA (13). Catalysis occurs through a ping-pong mechanism that is initiated when the active site cysteine residue in Sa SrtA nucleophilically attacks the backbone carbonyl carbon of the threonine residue within the LPXTG motif, breaking the threonine-glycine peptide bond to create a sortase-protein complex in which the components are linked via a thioacyl bond (14,15). The protein is then transferred by Sa SrtA to the cell wall precursor lipid II, when the amino group in this molecule nucleophilically attacks the thioacyl linkage to create a peptide bond-linked protein-lipid II product.…”