title: Elucidation of the protonation states of the catalytic residues in mtKasA: implications for inhibitor design.
abstract: KasA (β-ketoacyl ACP synthase I) is involved in the biosynthetic pathway of mycolic acids, an essential component of the cell wall in Mycobacterium tuberculosis. It was shown that KasA is essential for the survival of the pathogen and thus could serve as a new drug target for the treatment of tuberculosis. The active site of KasA was previously characterized by X-ray crystallography. However, questions regarding the protonation state of specific amino acids, the orientation of the histidine groups within the active site, and additional conformers being accessible at ambient temperatures remain open and have to be addressed prior to the design of new inhibitors. We investigate the active site of KasA in this work by means of structural motifs and relative energies. Molecular dynamics (MD) simulations, free energy perturbation computations, and calculations employing the hybrid quantum mechanics/molecular mechanics (QM/MM) method made it possible to determine the protonation status and reveal important details about the catalytic mechanism of KasA. Additionally, we can rationalize the molecular basis for the acyl-transfer activity in the H311A mutant. Our data strongly suggest that inhibitors should be able to inhibit different protonation states because the enzyme can switch easily between a zwitterionic and neutral state.
authors: Institut für Physikalische und Theoretische Chemie, Universität Würzburg, Am Hubland, D-97070 Würzburg, Germany.
authors: Wook
authors: W
authors: Lee
authors: Sylvia R
authors: SR
authors: Luckner
authors: Caroline
authors: C
authors: Kisker
authors: Peter J
authors: PJ
authors: Tonge
authors: Bernd
authors: B
authors: Engels
references: J Bacteriol. 2005 Nov;187(22):7596-606
references: 16267284
references: J Comput Chem. 2005 Dec;26(16):1781-802
references: 16222654
references: FEBS J. 2006 Feb;273(4):695-710
references: 16441657
references: J Bacteriol. 2003 Jul;185(14):4136-43
references: 12837788
references: Proteins. 1997 Jan;27(1):9-25
references: 9037708
references: EMBO J. 1998 Mar 2;17(5):1183-91
references: 9482715
references: Proteins. 2006 Nov 15;65(3):712-25
references: 16981200
references: J Mol Biol. 2001 Jul 6;310(2):449-70
references: 11428900
references: J Mol Graph. 1996 Jun;14(3):136-41
references: 8901641
references: Biochemistry. 2001 Aug 21;40(33):9836-45
references: 11502177
references: Cell. 2001 Feb 23;104(4):477-85
references: 11239406
references: J Biol Chem. 2000 Jun 2;275(22):16857-64
references: 10747933
references: Biomacromolecules. 2004 May-Jun;5(3):1066-77
references: 15132701
references: Annu Rev Biochem. 2005;74:791-831
references: 15952903
references: J Comput Chem. 2004 Aug;25(11):1400-15
references: 15185334
references: Mol Microbiol. 2007 Jun;64(6):1442-54
references: 17555433
references: Structure. 2001 Mar 7;9(3):233-43
references: 11286890
references: J Comput Chem. 2008 May;29(7):1019-31
references: 18072177
references: Structure. 2009 Jul 15;17(7):1004-13
references: 19604480
references: Annu Rev Microbiol. 2001;55:305-32
references: 11544358
references: Biochem Cell Biol. 2004 Feb;82(1):145-55
references: 15052334
references: J Mol Biol. 2001 Jan 19;305(3):491-503
references: 11152607
references: Biochemistry. 1991 Jun 18;30(24):5826-32
references: 2043624
references: Antimicrob Agents Chemother. 1996 Dec;40(12):2813-9
references: 9124847
references: FEMS Immunol Med Microbiol. 2008 Jul;53(2):145-50
references: 18479439
references: J Phys Chem B. 1998 Apr 30;102(18):3586-616
references: 24889800
references: Prog Lipid Res. 2003 Jul;42(4):289-317
references: 12689621
references: Mol Cell. 2000 Apr;5(4):717-27
references: 10882107
references: Mol Microbiol. 2000 May;36(3):630-7
references: 10844652
references: J Am Chem Soc. 2008 Jul 9;130(27):8696-705
references: 18557615
references: Biochem J. 2002 Jun 1;364(Pt 2):423-30
references: 12023885
references: Annu Rev Med. 2009;60:307-20
references: 19630575
references: J Comput Chem. 2006 Jul 30;27(10):1101-11
references: 16691568
references: J Biol Chem. 2006 Jun 23;281(25):17390-17399
references: 16618705