Studying aminoglycoside modification by the acetyltransferase class of resistance-causing enzymes via microarray.
Aminoglycosides are broad-spectrum antibacterials to which some bacteria have acquired resistance. The most common mode of resistance to aminoglycosides is enzymatic modification of the drug by different classes of enzymes including acetyltransferases (AACs). Thus, the modification of aminoglycosides by AAC(2') from Mycobacterium tuberculosis and AAC(3) from Escherichia coli was studied using aminoglycoside microarrays. Results show that both enzymes modify their substrates displayed on an array surface in a manner that mimics their relative levels of modification in solution. Because aminoglycosides that are modified by resistance-causing enzymes have reduced affinities for binding their therapeutic target, the bacterial rRNA aminoacyl-tRNA site (A-site), arrays were probed for binding to a fluorescently labeled oligonucleotide mimic of the A-site after modification. A decrease in binding was observed when aminoglycosides were modified by AAC(3). In contrast, a decrease in binding of the A-site is not observed when aminoglycosides are modified by AAC(2'). Interestingly, these effects mirror the biological functions of the enzymes: the AAC(3) used in this study is known to confer aminoglycoside resistance, while the AAC(2') is chromosomally encoded and unlikely to play a role in resistance. These studies lay a direct foundation for studying resistance to aminoglycosides and can also have more broad applications in identifying and studying non-aminoglycoside carbohydrates or proteins as substrates for acetyltransferase enzymes.
Barrett
Olivia J
OJ
Department of Chemistry, The Center for Excellence in Bioinformatics and Life Sciences, University at Buffalo, 657 Natural Sciences Complex, Buffalo, NY 14260, USA.
Pushechnikov
Alexei
A
Wu
Meilan
M
Disney
Matthew D
MD
D000123:Acetyltransferases / Q000378:metabolism*; D000617:Aminoglycosides / Q000737:chemistry / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000187:drug effects / Q000378:metabolism; D002236:Carbohydrate Conformation; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000201:enzymology; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000201:enzymology; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012329:RNA, Bacterial / Q000187:drug effects / Q000378:metabolism; D012335:RNA, Ribosomal / Q000187:drug effects / Q000378:metabolism; D013379:Substrate Specificity; D014031:Tobramycin / Q000494:pharmacology
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D000123:Acetyltransferases; D014031:Tobramycin
Science. 2000 Sep 8;289(5485):1760-3
10976071
Chem Biol. 2002 Apr;9(4):443-54
11983333
Nat Protoc. 2007;2(11):2747-58
18007610
Angew Chem Int Ed Engl. 2004 Mar 12;43(12):1591-4
15022242
Nat Genet. 2000 Nov;26(3):283-9
11062466
Chem Rev. 2005 Feb;105(2):477-98
15700953
Nat Biotechnol. 2002 Oct;20(10):1011-7
12219077
Biochemistry. 2007 Oct 9;46(40):11223-30
17867707
Bioorg Med Chem. 1999 Jul;7(7):1361-71
10465410
Nat Biotechnol. 2002 Mar;20(3):275-81
11875429
Biochemistry. 2005 Dec 13;44(49):16275-83
16331988
J Mol Biol. 1987 Feb 20;193(4):661-71
2441068
J Am Chem Soc. 2003 Jul 16;125(28):8420-1
12848532
Angew Chem Int Ed Engl. 2003 May 30;42(21):2376-9
12783500
ACS Chem Biol. 2007 Nov 20;2(11):745-54
17975888
Chemistry. 2004 Jul 5;10(13):3308-14
15224340
J Antibiot (Tokyo). 1982 May;35(5):574-9
7107522
Science. 1944 Aug 4;100(2588):103-5
17788929
Nucleic Acids Res. 2000 Jun 15;28(12):E66
10871389
Proc Natl Acad Sci U S A. 2004 Dec 7;101(49):17033-8
15563589
J Am Chem Soc. 2004 Mar 24;126(11):3447-53
15025471
Nature. 2002 Apr 11;416(6881):653-7
11948353
Proc Natl Acad Sci U S A. 1945 Jun;31(6):157-64
16578153
Science. 1995 Oct 20;270(5235):467-70
7569999
Glycobiology. 2004 Feb;14(2):197-203
14638630
J Am Chem Soc. 2006 Feb 1;128(4):1261-71
16433544
Angew Chem Int Ed Engl. 2004 Jun 14;43(24):3177-82
15199571
Nat Chem Biol. 2007 Sep;3(9):541-8
17710100
Chembiochem. 2007 Apr 16;8(6):649-56
17394189
J Biol Chem. 2001 Dec 7;276(49):45876-81
11590162
Nat Struct Biol. 2002 Sep;9(9):653-8
12161746
Science. 1972 Nov 24;178(4063):871-2
5085985
Nature. 1987 Jun 4-10;327(6121):389-94
2953976
Chem Biol. 2002 Apr;9(4):455-63
11983334
Chem Biol. 2004 Dec;11(12):1701-7
15610854
Chem Biol. 2002 Jun;9(6):747-55
12079787
Chem Biol. 2004 Apr;11(4):535-46
15123248
Science. 2001 May 4;292(5518):897-902
11340196
Org Lett. 2004 Aug 19;6(17):2853-5
15330631
Science. 1999 Sep 10;285(5434):1722-5
10481006
Antimicrob Agents Chemother. 2000 Jan;44(1):39-42
10602720
Angew Chem Int Ed Engl. 2002 Sep 2;41(17):3180-2
12207382
Nucleic Acids Res. 2005 Oct 07;33(17):5677-90
16214802
Science. 1996 Nov 22;274(5291):1367-71
8910275
Antimicrob Agents Chemother. 1978 Jul;14(1):69-72
356726
Chem Biol. 1998 Jul;5(7):397-406
9662506