Scabin, a Novel DNA-acting ADP-ribosyltransferase From Streptomyces Scabies

J Biol Chem. 2016 May 20;291(21):11198-215. doi: 10.1074/jbc.M115.707653. Epub 2016 Mar 21.

Abstract

A bioinformatics strategy was used to identify Scabin, a novel DNA-targeting enzyme from the plant pathogen 87.22 strain of Streptomyces scabies Scabin shares nearly 40% sequence identity with the Pierisin family of mono-ADP-ribosyltransferase toxins. Scabin was purified to homogeneity as a 22-kDa single-domain enzyme and was shown to possess high NAD(+)-glycohydrolase (Km (NAD) = 68 ± 3 μm; kcat = 94 ± 2 min(-1)) activity with an RSQXE motif; it was also shown to target deoxyguanosine and showed sigmoidal enzyme kinetics (K0.5(deoxyguanosine) = 302 ± 12 μm; kcat = 14 min(-1)). Mass spectrometry analysis revealed that Scabin labels the exocyclic amino group on guanine bases in either single-stranded or double-stranded DNA. Several small molecule inhibitors were identified, and the most potent compounds were found to inhibit the enzyme activity with Ki values ranging from 3 to 24 μm PJ34, a well known inhibitor of poly-ADP-ribosyltransferases, was shown to be the most potent inhibitor of Scabin. Scabin was crystallized, representing the first structure of a DNA-targeting mono-ADP-ribosyltransferase enzyme; the structures of the apo-form (1.45 Å) and with two inhibitors (P6-E, 1.4 Å; PJ34, 1.6 Å) were solved. These x-ray structures are also the first high resolution structures of the Pierisin subgroup of the mono-ADP-ribosyltransferase toxin family. A model of Scabin with its DNA substrate is also proposed.

Keywords: DNA binding protein; bacterial toxin; bioinformatics; crystallography; enzyme kinetics; fluorescence; molecular modeling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADP Ribose Transferases / antagonists & inhibitors
  • ADP Ribose Transferases / chemistry
  • ADP Ribose Transferases / metabolism*
  • Amino Acid Sequence
  • Bacterial Proteins / antagonists & inhibitors
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / metabolism*
  • Bacterial Toxins / chemistry
  • Crystallography, X-Ray
  • DNA, Bacterial / metabolism
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology
  • Kinetics
  • Molecular Dynamics Simulation
  • Sequence Homology, Amino Acid
  • Streptomyces / enzymology*
  • Streptomyces / genetics
  • Streptomyces / pathogenicity
  • Substrate Specificity

Substances

  • Bacterial Proteins
  • Bacterial Toxins
  • DNA, Bacterial
  • Enzyme Inhibitors
  • ADP Ribose Transferases

Associated data

  • PDB/2CB4
  • PDB/2CB6
  • PDB/5DAZ
  • PDB/5EWK
  • PDB/5EWY