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

Joydeep Ghosh1, Patricia J Anderson, Sukantha Chandrasekaran

  • 1Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

The Journal of Biological Chemistry
|December 19, 2009
PubMed

Insights

Streptococcus pyogenes beta-NAD(+) glycohydrolase (SPN) was investigated. Contrary to prior reports, SPN functions solely as a beta-NAD(+) hydrolase, not an ADP-ribosyl cyclase or transferase, impacting host cell death during infection.

Area of Science:

  • Microbiology
  • Enzymology
  • Pathogenesis

Background:

  • * Streptococcus pyogenes is a gram-positive pathogen that injects beta-NAD(+) glycohydrolase (SPN) into host cells.
  • * SPN's role in pathogenesis is linked to its enzymatic activities, potentially including beta-NAD(+) hydrolysis, ADP-ribosyl cyclase, and ADP-ribosyltransferase functions.
  • * Previous studies suggested SPN possessed multiple enzymatic activities, but its precise function remained unclear.

Purpose of the Study:

  • * To comprehensively characterize the enzymatic activities of purified recombinant SPN.
  • * To elucidate the specific mechanism by which SPN contributes to host cell death during Streptococcus pyogenes infection.
  • * To determine if SPN possesses ADP-ribosyl cyclase and ADP-ribosyltransferase activities in addition to its glycohydrolase function.

Main Methods:

  • * Production and purification of recombinant SPN.
  • * Kinetic analysis of SPN's enzymatic activities, including beta-NAD(+) hydrolysis, ADP-ribosyl cyclase, and ADP-ribosyltransferase assays.
  • * Investigation of product inhibition and substrate specificity.
  • * Functional analysis of SPN toxicity in Saccharomyces cerevisiae.

Main Results:

  • * Purified recombinant SPN exclusively exhibited beta-NAD(+) hydrolytic activity.
  • * SPN lacked detectable ADP-ribosyl cyclase and ADP-ribosyltransferase activities.
  • * Kinetic studies revealed an ordered uni-bi mechanism for SPN, with ADP-ribose released as the second product.
  • * SPN demonstrated toxicity in yeast, which was abolished by inactivating its glycohydrolase function.

Conclusions:

  • * SPN functions strictly as a beta-NAD(+) glycohydrolase during Streptococcus pyogenes pathogenesis.
  • * The previously reported cyclase and transferase activities of SPN were not confirmed in this study.
  • * SPN's beta-NAD(+) hydrolytic activity is the primary mechanism contributing to host cell death.

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