Purification and characterization of the single-strand-specific and guanylic-acid-preferential deoxyribonuclease activity of the extracellular nuclease from Basidiobolus haptosporus.
An extracellular nuclease from Basidiobolus haptosporus (designated as nuclease Bh1) was purified to homogeneity by ammonium sulfate precipitation, heat treatment, negative adsorption on DEAE-cellulose, and chromatography on phenyl-Sepharose followed by FPLC on phenyl-Superose. The overall yield was 26%. The Mr of the purified enzyme, determined by gel filtration, was 41 000 whereas by SDS/PAGE (after deglycosylation) it was 30 000. It is a glycoprotein with a pI of 6.8. The optimum pH and temperature for DNA hydrolysis were 8. 5 and 60 degrees C, respectively. Nuclease Bh1 is a metalloprotein but has no obligate requirement for metal ions to be active, nor is its activity stimulated in the presence of metal ions. The enzyme was inhibited by Zn2+, Ag2+, Hg2+, Fe3+ and Al3+, inorganic phosphate, pyrophosphate, dithiothreitol, 2-mercaptoethanol, NaCl and KCl. It was stable to high concentrations of organic solvents and urea but susceptible to low concentrations of SDS and guanidine hydrochloride. Nuclease Bh1 is a multifunctional enzyme and its substrate specificity is in the order of ssDNA approximately 3'AMP >> RNA > dsDNA. Studies on its mode of action showed that it cleaved supercoiled pUC 18 DNA and phage M13 DNA, endonucleolytically, generating single base nicks. The enzyme hydrolyzed DNA with preferential liberation of 5'dGMP, suggesting it to be a guanylic acid preferential endoexonuclease. 5'dGMP, the end product of hydrolysis, was a competitive inhibitor of the enzyme. The absence of 5'dCMP as a hydrolytic product, coupled with the resistance of (dC)10 and deoxyribodinucleoside monophosphates having cytosine either at the 3' or the 5' end, indicates that C-linkages are resistant to cleavage by nuclease Bh1.
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
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Desai
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Shankar
D000595:Amino Acid Sequence; D002412:Cations / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003851:Deoxyribonucleases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004720:Endonucleases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020091:Entomophthorales / Q000201:enzymology* / Q000254:growth & development; D004795:Enzyme Stability; D005092:Exonucleases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005656:Fungal Proteins; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
D002412:Cations; D005656:Fungal Proteins; C414304:Bh1 protein, Basidiobolus haptosporus; D003851:Deoxyribonucleases; D004720:Endonucleases; D005092:Exonucleases