We have determined the solution NMR structure of SACOL2532, a putative GCN5-like N-acetyltransferase (GNAT) from Staphylococcus aureus. SACOL2532 was shown to bind both CoA and acetyl-CoA, and structures with and without bound CoA were determined. Based on analysis of the structure and sequence, a subfamily of small GCN5-related N-acetyltransferase (GNAT)-like proteins can be defined. Proteins from this subfamily, which is largely congruent with COG2388, are characterized by a cysteine residue in the acetyl-CoA binding site near the acetyl group, by their small size in relation to other GNATs, by a lack of obvious substrate binding site, and by a distinct conformation of bound CoA in relation to other GNATs. Subfamily members are found in many bacterial and eukaryotic genomes, and in some archaeal genomes. Whereas other GNATs transfer the acetyl group of acetyl-CoA directly to an aliphatic amine, the presence of the conserved cysteine residue suggests that proteins in the COG2388 GNAT-subfamily transfer an acetyl group from acetyl-CoA to one or more presently unidentified aliphatic amines via an acetyl (cysteine) enzyme intermediate. The apparent absence of a substrate-binding region suggests that the substrate is a macromolecule, such as another protein, or that a second protein subunit providing a substrate-binding region must combine with SACOL2532 to make a fully functional N-acetyl transferase.
Acetyl coenzyme A Acetyl enzyme Acyl enzyme GCN5 GNAT N-acetyltransferase
Heteronuclear multiple quantum coherence
Heteronuclear single quantum coherence
Nuclear magnetic resonance
Nuclear Overhauser effect (spectroscopy)
Root mean square deviation
This is a preview of subscription content, log in to check access.
This research was supported by a grant from the Protein Structure Initiative of the National Institutes of Health (U54 GM074958). NMR spectra were acquired in the Environmental Molecular Sciences Laboratory (a national scientific user facility sponsored by the U.S. Department of Energy Office of Biological and Environmental Research) located at Pacific Northwest National Laboratory and operated for DOE by Battelle (contract KP130103). We thank Luciano Mueller for assistance with the doubly-filtered 1H–1H NOESY and TOCSY experiments.
Andres HH, Klem AJ, Schopfer LM, Harrison JK, Weber WW (1988) On the active-site of liver acetyl-CoA-arylamine N-acetyltransferase from rapid acetylator rabbits (III/J). J Biol Chem 263:7521–7527PubMedGoogle Scholar
Gill SR, Fouts DE, Archer GL, Mongodin EF, Deboy RT, Ravel J et al (2005) Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producting methicillin-resistant Staphylococcus epidermis strain. J Bacteriol 187:2426–2438. doi:10.1128/JB.187.7.2426-2438.2005PubMedCrossRefGoogle Scholar
Huang YJ, Powers R, Montelione GT (2005) Protein NMR recall, precision, and F-measure scores (RPF scores): structure quality assessment measures based on information retrieval statistics. J Am Chem Soc 127:1665–1674. doi:10.1021/ja047109hPubMedCrossRefGoogle Scholar
Huang YJ, Tejero R, Powers R, Montelione GT (2006) AutoStructure: a topology-constrained distance network algorithm for protein structure determination from NOESY data. Proteins 62:587–603. doi:10.1002/prot.20820PubMedCrossRefGoogle Scholar
Laskowski RA, Rullmann JAC, MacArthur MW, Kaptein R, Thornton JM (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR 8:477–486. doi:10.1007/BF00228148PubMedCrossRefGoogle Scholar
Lin Y, Fletcher CM, Zhou J, Allis CD, Wagner G (1999) Solution structure of the catalytic domain of GCN5 histone acetyltransferase bound to coenzyme A. Nature 400:86–89. doi:10.1038/21922PubMedCrossRefGoogle Scholar
Neri D, Szyperski T, Otting G, Senn H, Wütrich K (1989) Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling. Biochemistry 28:7510–7516. doi:10.1021/bi00445a003PubMedCrossRefGoogle Scholar
Tatusov RL, Natale DA, Garkavtsev IV, Tatusova TA, Shankavaram UT, Rao BS et al (2001) The COG database: new developments in phylogenetic classification of proteins from complete genomes. Nucleic Acids Res 29:22–28. doi:10.1093/nar/29.1.22PubMedCrossRefGoogle Scholar
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680. doi:10.1093/nar/22.22.4673PubMedCrossRefGoogle Scholar
Tyler RC, Bitto E, Berndsen CE, Bingman CA, Singh S, Lee MS et al (2006) Structure of Arabidopsis thaliana At1g77540 protein, a minimal acetyltransferase from the COG2388 family. Biochemistry 45:14325–14336. doi:10.1021/bi0612059PubMedCrossRefGoogle Scholar
Vuister GW, Bax A (1993) Quantitative J correlation: a new approach for measuring homonuclear three-bond J(HNHα) coupling constatnts in 15N-enriched proteins. J Am Chem Soc 115:7772–7777. doi:10.1021/ja00070a024CrossRefGoogle Scholar
Zwahlen C, Legault P, Vincent SJF, Greenblatt J, Konrat R, Kay LE (1997) Methods for measurement of intermolecular NOEs by multinuclear NMR spectroscopy: application to a bacteriophage λN-peptide/boxB RNA complex. J Am Chem Soc 119:6711–6721. doi:10.1021/ja970224qCrossRefGoogle Scholar