Properties of the major non-specific endonuclease from the strict anaerobe Fibrobacter succinogenes and evidence for disulfide bond formation in vivo.
DNase A is a non-specific endonuclease of Fibrobacter succinogenes. The enzyme was purified to homogeneity and its properties studied both in vitro and in vivo. Magnesium but not calcium was essential for nucleolytic activity. Manganese ions substituted for magnesium but were less stimulatory. DNase A activity was markedly inhibited by either NaCl or KCl at concentrations greater than 75 mM. The enzyme had a temperature optimum of 25 degrees C and a pH optimum of about 7.0. Values for K:(m) and K:(cat) were determined to be 61 microM and 330 s(-1) respectively, with a catalytic efficiency approximately threefold greater than bovine pancreatic DNase I, but 10-fold less than the Serratia marcescens NucA. DNase A was localized to the periplasm and probably exists as a monomeric species. The enzyme possessed one or more disulfide bonds. In the reduced form it had an apparent mass of 33 kDa, while in the oxidized form it was 29 kDa as estimated by SDS-PAGE. Reduction of the disulfide bonds by dithiothreitol with or without subsequent alkylation by iodoacetamide strongly inactivated the enzyme. DNase A accumulated in vivo had an apparent mass of 29 kDa, indicating that it was in an oxidized form. This is the first indication in a strict anaerobe of a functional periplasmic disulfide bond forming system, phenotypically similar to Dsb systems in facultative and aerobic bacteria.
MacLellan
Shawn R
SR
Department of Microbiology, University of Guelph, Guelph, Ontario, CanadaN1G 2W11.
Forsberg
Cecil W
CW
Department of Microbiology, University of Guelph, Guelph, Ontario, CanadaN1G 2W11.
D000818:Animals; D001421:Bacteria, Anaerobic / Q000201:enzymology* / Q000254:growth & development; D002417:Cattle; D003851:Deoxyribonucleases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004720:Endonucleases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012417:Rumen / Q000382:microbiology*; D013347:Subcellular Fractions / Q000378:metabolism
D004220:Disulfides; D003851:Deoxyribonucleases; D004720:Endonucleases; C019328:deoxyribonuclease A