Rapid purification and characterization of two distinct N-deoxyribosyltransferases of Lactobacillus leichmannii.
Two distinct N-deoxyribosyltransferases of Lactobacillus leichmannii, designated as DRTase I and DRTase II, were separated and purified almost to homogeneity by one-step affinity chromatography. DRTase I is distinguished by specifically catalyzing the direct transfer of 2-deoxyribosyl residues from purine deoxyribonucleosides to free purine bases, whereas DRTase II has a rather broad substrate specificity and is able to transfer the deoxyribosyl moiety between pyrimidines and between purines and pyrimidines. Furthermore, in addition to the different substrate spectrum, we clearly differentiated the two enzymes by comparing their varying temperature/activity and pH/activity profiles, their kinetic constants, their behaviour in Western blot analysis, and their N-terminal amino acid sequences. Denaturing and non-denaturing DISK-PAGE revealed strong evidence that both intact enzymes consist of hexamers with subunit molecular weights of approximately 20,000 for DRTase I and 18,000 for DRTase II.
Becker
J
J
Institut für Mikrobiologie, Johann Wolfgang Goethe-Universität, Frankfurt/Main, Germany.
Brendel
M
M
D000225:Adenine / Q000378:metabolism; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006147:Guanine / Q000378:metabolism; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification*; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010430:Pentosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D011489:Protein Denaturation; D013696:Temperature
D007527:Isoenzymes; D006147:Guanine; D010430:Pentosyltransferases; C021888:nucleoside deoxyribosyltransferase; D000225:Adenine