Purification and some properties of streptococcal NAD-glycohydrolase

Gerlach, Dieter; Ozegowski, Jörg‐Hermann; Günther, Elisabeth; Vettermann, Stefan; Khöler, W.

doi:10.1111/j.1574-6968.1996.tb08027.x

Cited by 16 publications

(18 citation statements)

References 18 publications

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“…The kinetic parameters for the ␤-NAD ϩ glycohydrolase reaction were determined using a sensitive HPLCbased assay (Table 1). The observed turnover number for SPN (Table 1) was similar to that determined for a related ␤-NAD ϩ glycohydrolase purified from a group C streptococcal species (12,000 s Ϫ1 ) (35). Comparison of this value to the reported k cat for the ADP-ribosyltransferase diphtheria toxin fragment A (0.0083 s Ϫ1 ) (33) shows the turnover number for SPN is ϳ1 ϫ 10 7 -fold higher, suggesting the activity may resemble that of the strict ␤-NAD ϩ glycohydrolases and not that of ADP-ribosyltransferases.…”

Section: Kinetic Parameters For Spn Hydrolysis Of ␤-Nadsupporting

confidence: 75%

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Characterization of Streptococcus pyogenes β-NAD+ Glycohydrolase

Ghosh

Anderson

Chandrasekaran

et al. 2010

Journal of Biological Chemistry

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The Gram-positive pathogen Streptococcus pyogenes injects a ␤-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 ␤-NAD ؉ -dependent enzyme activities. SPN has been reported to possess the unique characteristic of not only catalyzing hydrolysis of ␤-NAD ؉ , but also carrying out ADP-ribosyl cyclase and ADP- The enzymes that cleave the nicotinamide-ribosyl bond of ␤-NAD ϩ to produce numerous small molecules are able to modulate various aspects of cellular function, including signal transduction, vascular activity, gene expression, calcium homeostasis, and cell death (reviewed in Refs. 1, 2). All of these enzymes hydrolyze ␤-NAD ϩ to produce nicotinamide and adenosine diphosphoribose (ADPR) 3 (see Fig. 1 and Table 2). Interestingly, most enzymes within this class are multifunctional and can be further classified on the basis of the additional reactions that they can catalyze following the release of nicotinamide (see Fig. 1 and Table 2). The ADP-ribosyl cyclases (EC 3.2.2.6) convert enzyme-bound ADPR to cyclic ADPR (cADPR) (reviewed in Refs. Only the strict ␤-NAD ϩ glycohydrolases (EC 3.2.2.5) are not capable of further catalysis of the products of the initial reaction, and, as a general rule, the cyclases and transferases do not catalyze each other's reactions (Fig. 1).An exception to the above rule, the Streptococcus pyogenes ␤-NAD ϩ glycohydrolase (SPN, also known as Nga) is the only enzyme reported to possess all three activities (6 -9). These activities can contribute to S. pyogenes virulence as, after SPN is exported, the enzyme is injected across the host cell membrane into the cytosol by a process called cytolysin-mediated translocation (10, 11). Once in the cytosol, SPN likely contributes to the pathogenesis of the numerous different diseases that S. pyogenes can cause, including pharyngitis, impetigo, necrotizing faciitis rheumatic fever, or acute glomerulonephritis (12). The contribution of SPN to pathogenesis has been demonstrated in several model systems. For example, mutants of S. pyogenes engineered to lack SPN were avirulent in animal models of streptococcal infection (13), and cytosolic SPN was highly cytotoxic to yeast and to cultured epithelial cells (10,11,14). However, the mechanism for pathogenesis by SPN remains unknown. Given the number and diversity of the different diseases that S. pyogenes can cause, an understanding of the contribution of SPN to pathogenesis of any streptococcal disease requires a detailed characterization of its kinetic and catalytic properties.Until recently, characterization of the kinetic properties of SPN has been hindered due to difficulties in expression of recombinant SPN owing to its toxicity. Toxicity is so severe that expression plasmids containing the gene for SPN cannot be * This work was supported, in whole or in part, by National Institutes of Health Grant AI064721 (to M. G. C.)...

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Section: Kinetic Parameters For Spn Hydrolysis Of ␤-Nadsupporting

confidence: 75%

“…7). This was an unexpected result, because the ␤-NAD ϩ glycohydrolases from the related group C streptococcal species do not hydrolyze ␤-NADP ϩ (35). However, analysis of Michaelis-Menten parameters revealed that NADP ϩ is a poor substrate for SPN with an apparent K m of 1.7 Ϯ 0.4 mM and observed k cat of 384 Ϯ 56 s Ϫ1 .…”

Section: Spn Lacks ␤-Nadmentioning

confidence: 96%

Characterization of Streptococcus pyogenes β-NAD+ Glycohydrolase

Ghosh

Anderson

Chandrasekaran

et al. 2010

Journal of Biological Chemistry

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“…Genetic and functional investigations of these inhibitors have not yet been performed. On the other hand, hemolytic streptococci secrete thermolabile NADase into culture medium (27)(28)(29). Streptococcal cells producing the extracellular NADase contain an inhibitor protein against this enzyme (30).…”

Section: Discussionmentioning

confidence: 99%

“…NADase Assay-The NADase assay was performed by using the method of Gerlach et al (29). One unit of the enzyme cleaves 1 mol of NAD in 1 min at 37°C.…”

Section: Purification Of His Tag-fused Orf1-the Bugbuster Extract Of mentioning

confidence: 99%

Genetic and Biochemical Properties of Streptococcal NAD-glycohydrolase Inhibitor

Kimoto

Fujii

Hirano³

et al. 2006

Journal of Biological Chemistry

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The gene encoding streptolysin O (slo), a cytolysin of hemolytic streptococci, is transcribed polycistronically from the promoter of the preceding NAD-glycohydrolase (NADase) gene (nga). Between nga and slo, a putative open reading frame (orf1) is located whose function has been totally unknown. Present investigation demonstrated that the orf1 encodes a protein designated as streptococcal NADase inhibitor (SNI). From its nucleotide sequence, SNI was inferred to comprise 161 amino acid residues and the deduced molecular weight was 18,800. This protein was detectable only within cells. Coexpression of SNI was essential for production of streptococcal NADase, and NADase precursor existed as an inactive complex with SNI, in recombinant Escherichia coli. Monomeric NADase and SNI rapidly formed in vitro a stable heterodimer complex in the ratio 1:1, resulting in complete suppression of the hydrolase activity. Unlike other bacterial NADase inhibitors, SNI was thermostable. This protein, coexpressed and complexed with NADase, may protect the producer cocci from exhaustion of NAD.Besides the Lancefield group A (Streptococcus pyogenes, GAS), 3 human isolates of group C (GCS, including Streptococcus dysgalactiae subsp. equisimilis) and G (GGS) streptococci have been implicated as causative agents in outbreaks of purulent pharyngitis, wound infections, and streptococcal toxic shock-like syndrome (1-3). Streptolysin O (SLO), an oxygen-labile cholesterol-dependent cytolysin, is an extracellular protein produced by most strains of GAS and many strains of GCS and GGS (4). SLO belongs to a single, highly homologous family of cytolysins found in species of five genera: Streptococcus, Bacillus, Clostridium, Listeria, and Arcanobacterium (5-10). Upon binding to cholesterol-containing membrane as monomers, these proteins oligomerize to form large pores, comprising ϳ50 monomer subunits with internal diameter of up to 30 nm (9, 10). These large pores permit permeation of not only ions and small metabolites but also macromolecules. Madden et al. (11) proposed, for the first time from Gram-positive bacteria, a cytolysin-mediated translocation system that is a functional equivalent to the type III secretion system in Gram-negative bacteria (12)(13)(14)(15). Their data support a model in which the effector NAD-glycohydrolase (NADase), encoded by nga (spn) located upstream of the slo gene, is transported through the SLO pore into the host cell (11). Thus SLO is of considerable biochemical and clinical interest, and elucidation of expression mechanism of slo gene is important for understanding streptococcal pathogenesis as well.Recently, we demonstrated that slo is a member of an operon covering the upper-stream nusG and nga genes (nusG-nga-orf1-slo), from which transcription of slo proceeds polycistronically, and major transcript is produced by readthrough from nga promoter (16). The arrangement commonly conserved in streptococci was limited to the region from nusG to slo (17-21). These results suggest intimate relationship among NADase, ...

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“…NAD-glycohydrolase was previously puri¢ed from group C streptococci and its N-terminal sequence determined [18]. This sequence (40 amino acids) was used to search the S. pyogenes strain SF370 genome database (www.genome.ou.edu).…”

Section: Identi¢cation Of the Gene That Encodes Nad-glycohydrolase Ofmentioning

confidence: 99%

The NAD-glycohydrolase (nga) gene of Streptococcus pyogenes

Ajdic¹

2000

FEMS Microbiology Letters

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The gene for NAD-glycohydrolase (nga) of group A streptococci (Streptococcus pyogenes) was identified and shown to be located immediately adjacent to the gene for streptolysin O (slo). The nga gene contains 1341 base pairs and encodes a protein of 447 amino acids, including an N-terminal signal peptide. Results from analysis with the polymerase chain reaction indicated that the nga gene is present in all of the strains tested. Functional extracellular NAD-glycohydrolase, also known as NADase, was detected among a wide variety of clinical isolates and known laboratory strains and shown to be present in 72% of 100 strains examined. In contrast, 92% of strains isolated from patients with invasive streptococcal infections were positive for NADase production. ß

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Purification and some properties of streptococcal NAD-glycohydrolase

Cited by 16 publications

References 18 publications

Characterization of Streptococcus pyogenes β-NAD+ Glycohydrolase

Characterization of Streptococcus pyogenes β-NAD+ Glycohydrolase

Genetic and Biochemical Properties of Streptococcal NAD-glycohydrolase Inhibitor

The NAD-glycohydrolase (nga) gene of Streptococcus pyogenes

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