Characterization of Streptococcus pyogenes beta-NAD+ glycohydrolase: re-evaluation of enzymatic properties associated with pathogenesis

Abstract

The gram-positive pathogen Streptococcus pyogenes injects a beta-NAD(+) glycohydrolase (SPN) into the cytosol of an infected host cell using the cytolysin-mediated translocation pathway. In this compartment, SPN accelerates the death of the host cell by an unknown mechanism that may involve its beta-NAD(+)-dependent enzyme activities. SPN has been reported to possess the unique characteristic of not only catalyzing hydrolysis of beta-NAD(+), but also carrying out ADP-ribosyl cyclase and ADP-ribosyltransferase activities, making SPN the only beta-NAD(+) glycohydrolase that can catalyze all of these reactions. With the long term goal of understanding how these activities may contribute to pathogenesis, we have further characterized the enzymatic activity of SPN using highly purified recombinant protein. Kinetic studies of the multiple activities of SPN revealed that SPN possessed only beta-NAD(+) hydrolytic activity and lacked detectable ADP-ribosyl cyclase and ADP-ribosyltransferase activities. Similarly, SPN was unable to catalyze cyclic ADPR hydrolysis, and could not catalyze methanolysis or transglycosidation. Kinetic analysis of product inhibition by recombinant SPN demonstrated an ordered uni-bi mechanism, with ADP-ribose being released as a second product. SPN was unaffected by product inhibition using nicotinamide, suggesting that this moiety contributes little to the binding energy of the substrate. Upon transformation, SPN was toxic to Saccharomyces cerevisiae, whereas a glycohydrolase-inactive SPN allowed for viability. Taken together, these data suggest that SPN functions exclusively as a strict beta-NAD(+) glycohydrolase during pathogenesis.

FIGURE 1.

Different classes of β-NAD⁺ glycohydrolases. Enzymes that hydrolytically cleave the nicotinamide-ribosyl bond of β-NAD⁺ (shown in red) to form nicotinamide (blue) and ADPR (green and black) are known as β-NAD⁺ glycohydrolases and are further classified by the additional bonds (if any) whose formation they can catalyze (also shown in red). These include: A, covalent addition of ADPR to a specific substrate (mono-ADP-ribosyltransferases); B, cyclization of ADPR (ADP-ribosyl cyclases); or C, no additional reaction (strict β-NAD⁺ glycohydrolases). Thin lines indicate other reactions catalyzed by the ADP-ribosyl cyclases. The thin dashed line for the cADPR hydrolase reaction indicates that the cADPR is not a sequential intermediate during the hydrolytic conversion of β-NAD⁺ to ADPR. *, β to α inversion of the ribosyl linkage after the transfer of ADPR (21, 34).

FIGURE 2.

SPN has homology with both ADP-ribosyl cyclases and ADP-ribosyltransferases. The carboxyl-terminal enzymatic domain of SPN (residues 234–451) shares some homology with the enzymatic domains of eukaryotic ADP-ribosyl cyclase (A), and prokaryotic ADP-ribosyltransferases (B). Alignment of the catalytic glutamic acid residue of the ADP-ribosyl cyclases is the highlighted box and open arrow in A. Additional glutamic acid residues of SPN subjected to mutagenesis are indicated by the open arrows (A). The putative ARTT domain of SPN is highlighted by the bar over the sequence, and the putative catalytic glutamic acid residue and second glutamic acid/glutamine residue are shown by the closed arrows (A). A comparison of the putative ARTT motif of SPN to those characterized ADP-ribosyltransferases (24) is shown (B). The catalytic and associated glutamic acid/glutamine residues are highlighted. The two “turns” of the ARTT motif (5) are indicated (T1 and T2). Alignments (A) were generated using ClustalW (45).

FIGURE 3.

SPN has a functional ARTT motif. Mutant versions of SPN were constructed to change specific glutamic acid residues (E) of the putative “cyclase” or “ARTT” motifs highlighted in Fig. 3 to glycine (G) or glutamine (Q) residues, as indicated. Mutant proteins were expressed in S. pyogenes SPN1, and culture supernatants were analyzed for β-NAD⁺ glycohydrolase activity relative to wild-type JRS4 SPN (WT), as shown. Data presented represents the mean and standard error of the mean derived from three independent experiments.

FIGURE 4.

SPN is insensitive to free nicotinamide and competitively inhibited by ADP-ribose. SPN hydrolysis of β-NAD⁺ was subjected to product inhibition to determine further classify the enzyme. In the presence of increasing concentrations of nicotinamide (A), SPN activity was unaffected. Increasing concentrations of ADP-ribose (B) inhibited the reaction in a competitive manner, indicative of an ordered uni-bi mechanism. As there was little or no inhibition seen with increasing concentrations of nicotinamide, the data in the absence of the nicotinamide was fitted to the Michaelis-Menten equation with parameters described in the text. Analysis of the ADP-ribose inhibition data were fit as described under “Experimental Procedures” with the parameters in the text. Results were also fitted to the reciprocal of the equation under “Experimental Procedures” to obtain a Lineweaver-Burk plot (inset).

FIGURE 5.

SPN lacks cADPR hydrolysis and cyclase activity. A, analysis of cADPR hydrolase activity. Shown are HPLC chromatograms of the products generated by incubation of the various proteins indicated in the Fig. with 1 mm cADPR for 1 h (recombinant CD38) or for 6 h (all other proteins shown). Locations of the peaks representing cADPR and ADPR are shown at the top. Note that commercially obtained cADPR contained some ADPR, as shown in the chromatogram lacking enzyme. B, cycling assay to measure cADPR synthesis. Shown are plots of resorufin fluorescence over time generated from the indicated standard concentrations of cADPR used as reference standards (cADPR Stds) or from cADPR synthesized by enzymatic reactions conducted with the proteins listed in the Fig. Data shown are from a single experiment representative of at least three independent experiments with identical results.

FIGURE 6.

SPN lacks methanolysis activity. Presented are HPLC chromatograms showing the products obtained from non-enzymatic methanolysis of β-NAD⁺ or from reactions with the various proteins indicated. The identities of the various peaks are noted at the top. Data shown are from a single experiment representative of at least three independent experiments with identical results.

FIGURE 7.

SPN is deficient in transglycosidase activity toward NADP⁺ but is capable of hydrolysis. Shown are HPLC chromatograms of the reaction products obtained from incubation of 1 mm NAD(P)⁺ and 20 mm nicotinic acid with the various proteins indicated. The identities of the various peaks are noted at the top. Of note, the commercially available nicotinic acid contained low levels of nicotinic acid adenine dinucleotide phosphate (NAADP⁺). Data shown are from a single experiment representative of at least three independent experiments with identical results.

FIGURE 8.

SPN does not ADP-ribosylate proteins, but β-NAD⁺ glycohydrolase activity is required for cytotoxicity. A, presented is an autoradiogram of an SDS-PAGE analysis of the reactions conducted with the various proteins indicated or with no added protein (none), ³²P-β-NAD⁺, and either the presence (+) or absence (−) of a whole cell extract prepared from HeLa cells (Extract). JRS4 and HSC5 are recombinant SPN generated from naturally occurring β-NAD⁺ glycohydrolase-positive and -negative alleles, respectively. The migration of the protein molecular mass (kDa) standards is shown on the left while the migration of the ADP-ribosylated elongation factor-2 is indicated by the filled arrowhead on the right. The two open arrowheads show proteins ADP-ribosylated by an endogenous ADP-ribosyltransferase activity present in the HeLa whole cell extract. Data shown are from a single experiment representative of at least three independent experiments with identical results. B, shown is a Western blot analysis of whole cell extracts prepared from S. cerevisiae yeast expressing SPN that lacks β-NAD⁺ glycohydrolase activity (SPN[NADase⁻]) or the plasmid vector alone (vector). SPN is expressed from the pYES2 vector under a galactose-inducible promoter. Expression was assessed at the times indicated (in hours) following growth in the presence of 2% glucose (Glc) or 2% galactose (Gal). C, growth of the indicated strains in the presence of 2% galactose.