Purification and characterization of Dnase A, the major endonuclease of Fibrobacter succinogenes S85

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MacLellan, Shawn Roderick
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University of Guelph

The DNase A protein of 'F. succinogenes' S85 may play a role in preventing DNA uptake by this ruminal bacterium. Based on zymographic analysis, this enzyme appears to be the only detectable non-specific endonuclease expressed by 'F. succinogenes'. Specific nuclease activity remains constant throughout all growth phases and nuclease activity is exhibited mainly within an extracytoplasmic extract from cells. DNase A was purified to homogeneity by a combination of ammonium sulphate precipitation and chromatographic methods. Kinetic parameters for DNase A were measured from the cleavage of herring testes DNA and the pure enzyme exhibited a KM of 61 ± 12 m M and a 'kcat' of 330 ± 25 s-1. DNase A therefore has a catalytic efficiency approximately three-fold greater than bovine pancreatic DNase I. The enzyme has a requirement for divalent cation co-factors. Magnesium at a concentration of 20 mM stimulates maximal activity and manganese can substitute for magnesium, but is less stimulatory. Calcium ions or monovalent cations do not stimulate activity. DNase A exhibits a temperature optimum of 25°C and a pH optimum of 7.0. The enzyme is strongly inactivated by diethylpyrocarbonate, but not by iodoacetamide or tetranitromethane. Activity can be partially recovered from acylated enzyme after treatment with hydroxylamine-HCl. DNase A possesses one or more disulfide bonds and the protein is inactivated in the presence of reducing agents with or without subsequent alkylation by iodoacetamide. DNase A accumulates as an oxidized species ' in vivo'. An extracytoplasmic location for the active species may allow the enzyme to intercept and degrade DNA that has passed through the outer membrane. (Abstract shortened by UMI.)

DNase A, Fibrobacter succinogenes S85, DNA uptake, ruminal bacterium