title: Functional annotation of putative aminoglycoside antibiotic modifying proteins in Mycobacterium tuberculosis H37Rv.
abstract: The growing availability of sequences of bacterial genomes has revealed a number of open reading frames predicted by sequence alignment to encode antibiotic resistance proteins. The presence of these putative resistance genes within bacterial genomes raises important questions regarding potential reservoirs of resistance elements and their evolution. Here we examine four gene products encoding predicted aminoglycoside-aminocyclitol antibiotic modifying enzymes, two phosphotransferases and two acetyltransferases, derived from analysis of the genome sequence of Mycobacterium tuberculosis strain H37Rv with the goal of assigning biochemical function by purification of each protein and characterization of their ability to modify aminoglycoside antibiotics. Only one of these enzymes, the previously characterized aminoglycoside acetyltransferase AAC(2')-Ic, displayed compelling aminoglycoside modifying activity. While the putative phosphotransferase encoded by the Rv3225c gene did display low levels of aminoglycoside kinase activity, the predicted kinase encoded by the Rv3817 gene lacked any such activity. A potential aminoglycoside 6'-acetyltransferase, encoded by the Rv1347c gene, did not show antibiotic acylation activity but did demonstrate selective thioesterase activity with numerous acyl-CoAs. This activity, together with the genomic environment of the Rv1347c gene in a likely polyketide synthesis cluster, suggests a role for this protein in secondary metabolism and not in antibiotic modification. It was thus shown that only one of four putative aminoglycosides modifying enzymes derived from the whole genome sequencing of M. tuberculosis H37Rv showed sufficient predicted enzyme activity to be annotated as an aminoglycoside resistance element. This study demonstrates the necessity of biochemical annotation methods as a follow up to in silico sequence alignment-based methods of assigning gene product function.
authors.affiliation: Antimicrobial Research Centre, Department of Biochemistry, McMaster University, 1200 Main Street West, Hamilton, ON, Canada L8N 3Z5.
authors.forename: Kari-Ann
authors.initials: KA
authors.lastname: Draker
authors.forename: David D
authors.initials: DD
authors.lastname: Boehr
authors.forename: Nadine H
authors.initials: NH
authors.lastname: Elowe
authors.forename: Theresa J
authors.initials: TJ
authors.lastname: Noga
authors.forename: Gerard D
authors.initials: GD
authors.lastname: Wright
mesh_terms: D000123:Acetyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D002262:Carboxy-Lyases / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D016680:Genome, Bacterial; D008826:Microbial Sensitivity Tests; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D010770:Phosphotransferases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D000123:Acetyltransferases; C019681:aminoglycoside acetyltransferase; D010770:Phosphotransferases; D002262:Carboxy-Lyases; C021532:malonyl-CoA decarboxylase