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 °C and a pH optimum of about 7·0. Values for Km and Kcat were determined to be 61 μM 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.
AndersonJ. A. K., KuntzG. P. P., EvansH. H., SwiftS. J.1971; Preferential interaction of manganous ions with the guanine moiety in nucleosides, dinucleoside monophophates, and deoxyribonucleic acid. Biochemistry 10:4368–4374[CrossRef]
BaderM., MuseW., BallouD. P., GassnerC., BardwellJ. C. A.1999; Oxidative protein folding is driven by the electron transport system. Cell 98:217–227[CrossRef]
BradfordM. M.1976; A rapid and sensitive method for the quantitation of microgram amounts of protein using the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef]
ChessonA., ForsbergC. W. 1997; Polysaccharide degradation by rumen microorganisms. In The Rumen Microbial Ecosystem pp. 329–381Edited byHobsonP., StewartC. Andover: Chapman & Hall;
DawsonR. M., ElliotC. D. C., ElliotW. H., JonesK. M.1986; Vitamins and coenzymes. In Data for Biochemical Research pp. 122Edited byDawsonR. M. C., ElliotD. C., ElliotW. H., JonesK. M. Oxford: Clarendon Press;
DickieP., WeinerJ. H. 1979; Purification and characterization of membrane bound fumarate reductase from anaerobically grown Escherichia coli. Can J Biochem 57:813–821
DoherityA. J., WorrallA. F., ConnollyB. A.1995; The roles of arginine 41 and tyrosine 76 in the coupling of DNA recognition to phosphodiester bond cleavage by DNase I: a study using site-directed mutagenesis. J Mol Biol 251:366–377[CrossRef]
FriedhoffP., MeissG., KolmesB., PieperU., GimadutdinowO., UrbankeC., PingoudA.1996; Kinetic analysis of the cleavage of natural and synthetic substrates by the Serratia nuclease. Eur J Biochem 241:572–580[CrossRef]
GongJ., ForsbergC. W. 1993; Separation of outer and cytoplasmic membranes of Fibrobacter succinogenes and membrane and glycogen granule locations of glycanase and cellobiase. J Bacteriol 175:6810–6821
HaleS. P., PooleL. B., GerltJ. A.1993; Mechanism of the reaction catalyzed by staphylococcal nuclease: identification of the rate-determining step. Biochemistry 32:7479–7487[CrossRef]
HuangL., ForsbergC. W. 1988; Purification and comparison of the periplasmic and extracellular forms of the cellodextrinase from Bacteroides succinogenes. Appl Environ Microbiol 54:1488–1493
KejnovskyE., KyprJ.1998; Millimolar concentrations of zinc and other metal cations cause sedimentation of DNA. Nucleic Acids Res 26:5295–5299[CrossRef]
KobayashiT., ItoK.1999; Respiratory chain strongly oxidizes the CXXC motif of DsbB in the Escherichia coli disulfide bond formation pathway. EMBO J 18:1192–1198[CrossRef]
KunitzM.1950; Crystalline desoxyribonuclease. I. Isolation and general properties: spectrophotometric method for the measurement of desoxyribonuclease activity. J Gen Physiol 33:349–362[CrossRef]
KuoJ.1992; About non-linear regression. In SigmaPlot Scientific Graphics Software: Transformation and Curvefitting Reference pp. 7.1–7.2Edited byKuoJ. San Rafael, CA: Jandel Corporation;
LanglaisM., Tajmir-RiahiH. A., SavoieR.1990; Raman spectroscopic study of the effects of Ca2+, Mg2+, Zn2+ and Cd2+ ions on calf thymus DNA: binding sites and conformational changes. Biopolymers 30:743–752[CrossRef]
LeeS. F., ForsbergC. W., GibbinsA. M.1992; Type-II DNA restriction modification system and an endonuclease from the ruminal bacterium Fibrobacter succinogenes S85. J Bacteriol 174:5275–5283
MeinhardtS. W., GlassT. L.1994; Characterization of the NADH dehydrogenase and fumarate reductase of Fibrobacter succinogenes subsp. succinogenes S85. Arch Microbiol 162:329–334[CrossRef]
PooleL. B., LoveysD. A., HaleS. P., GerltJ. A., StanczyckS. M., BoltonP. H.1991; Deletion of the omega loop in the active site of staphylococcal nuclease: effect on catalysis and stability. Biochemistry 30:3621–3627[CrossRef]