Isolation from Nocardioides sp. strain CT16, purification, and characterization of a deoxycytidine deaminase extremely thermostable in the presence of D,L-dithiothreitol

Biosci Biotechnol Biochem. 2002 Aug;66(8):1646-51. doi: 10.1271/bbb.66.1646.

Abstract

A deoxycytidine deaminase that was extremely thermostable in the presence of dithiothreitol was found in a mesophilic bacterium isolated from soil. The bacterium was classified as a Nocardioides sp. The enzyme was purified to a homogeneous protein by treatment at 100 degrees C, ammonium sulfate precipitation, and chromatography on DEAE-Toyopearl, hydroxyapatite, and then Sephacryl S-100. Twenty micrograms of the pure enzyme was obtained from 811 mg of the starting crude protein. After treatment at 50 degrees C for 15 min in the absence of dithiothreitol, enzyme activity was 44% of the starting activity; after treatment at 100 degrees C for 2 h in the presence of 50 mm dithiothreitol, activity was 56% of the starting activity. Dithiothreitol greatly stabilized the enzyme. Activity was maximum at 99 degrees C. The Km values for deoxycytidine, cytidine, and methyl-deoxycytidine were 55.2, 140, and 130 microM, respectively. The molecular mass was estimated to be 52 kDa by gel permeation chromatography. The enzyme molecule was dissociated into two subunits each of 18 kDa subunit when reduced with mercaptoethanol.

MeSH terms

  • Chemical Precipitation
  • Chromatography, Gel
  • Cytidine / chemistry
  • Cytidine / metabolism
  • Cytidine Deaminase
  • Cytosine / chemistry
  • Cytosine / metabolism
  • Deoxycytidine Monophosphate / chemistry
  • Deoxycytidine Monophosphate / metabolism
  • Dithiothreitol / chemistry*
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Stability
  • Hot Temperature
  • Kinetics
  • Molecular Weight
  • Nocardiaceae / enzymology*
  • Nucleoside Deaminases / chemistry*
  • Nucleoside Deaminases / isolation & purification
  • Nucleoside Deaminases / metabolism
  • Substrate Specificity

Substances

  • Deoxycytidine Monophosphate
  • Cytidine
  • Cytosine
  • Nucleoside Deaminases
  • Cytidine Deaminase
  • deoxycytidine deaminase
  • Dithiothreitol