Functional cloning, heterologous expression, and purification of two different N-deoxyribosyltransferases from Lactobacillus helveticus.
Lactobacillus helveticus contains two types of N-deoxyribosyltransferases: DRTase I catalyzes the transfer of 2'-deoxyribose between purine bases exclusively whereas DRTase II is able to transfer the 2'-deoxyribose between two pyrimidine or between pyrimidine and purine bases. An Escherichia coli strain, auxotrophic for guanine and unable to use deoxyguanosine as source of guanine, was constructed to clone the corresponding genes. By screening a genomic bank for the production of guanine, the L. helveticus ptd and ntd genes coding for DRTase I and II, respectively, were isolated. Although the two genes have no sequence similarity, the two deduced polypeptides display 25.6% identity, with most of the residues involved in substrate binding and the active site nucleophile Glu-98 being conserved. Overexpression and purification of the two proteins shows that DRTase I is specific for purines with a preference for deoxyinosine (dI) > deoxyadenosine > deoxyguanosine as donor substrates whereas DRTase II has a strong preference for pyrimidines as donor substrates and purines as base acceptors. Purine analogues were substrates as acceptor bases for both enzymes. Comparison of DRTase I and DRTase II activities with dI as donor or hypoxanthine as acceptor and colocalization of the ptd and add genes suggest a specific role for DRTase I in the metabolism of dI.
Kaminski
Pierre Alexandre
PA
Unité de Chimie Organique, CNRS Unité de Recherche Associée 2128, Institut Pasteur, 25-28 rue du Dr. Roux, 75724 Paris cedex 15, France. akaminsk@pasteur.fr
D003001:Cloning, Molecular; D003849:Deoxyguanosine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D013058:Mass Spectrometry; D010430:Pentosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012641:Selection, Genetic; D013379:Substrate Specificity
D005964:Glucosyltransferases; D010430:Pentosyltransferases; C021888:nucleoside deoxyribosyltransferase; C420226:nucleoside deoxyribosyltransferase II; D003849:Deoxyguanosine