title: Characterisation of ATP-dependent Mur ligases involved in the biogenesis of cell wall peptidoglycan in Mycobacterium tuberculosis.
abstract: ATP-dependent Mur ligases (Mur synthetases) play essential roles in the biosynthesis of cell wall peptidoglycan (PG) as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw) operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery.
authors.affiliation: Department of Biological Sciences, Institute of Structural and Molecular Biology, Birkbeck, University of London, London, United Kingdom.
authors.forename: Tulika
authors.initials: T
authors.lastname: Munshi
authors.forename: Antima
authors.initials: A
authors.lastname: Gupta
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Evangelopoulos
authors.forename: Juan David
authors.initials: JD
authors.lastname: Guzman
authors.forename: Simon
authors.initials: S
authors.lastname: Gibbons
authors.forename: Nicholas H
authors.initials: NH
authors.lastname: Keep
authors.forename: Sanjib
authors.initials: S
authors.lastname: Bhakta
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism*; D010457:Peptidoglycan / Q000096:biosynthesis*; D010766:Phosphorylation; D011485:Protein Binding; D017510:Protein Folding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D010457:Peptidoglycan; D000255:Adenosine Triphosphate; C451020:PknA protein, Mycobacterium tuberculosis; C475060:PknB protein, Mycobacterium tuberculosis; D017346:Protein Serine-Threonine Kinases; D010453:Peptide Synthases; C554750:MurE protein, Mycobacterium tuberculosis; C053379:UDP-N-acetylmuramoylalanine-D-glutamate ligase
references.citation: J Antimicrob Chemother. 2009 Oct;64(4):774-81
references.pmid: 19656786
references.citation: Protein Expr Purif. 2001 Apr;21(3):393-400
references.pmid: 11281713
references.citation: Biochem Pharmacol. 2012 Sep 1;84(5):625-32
references.pmid: 22705647
references.citation: J Mol Biol. 2006 Sep 29;362(4):640-55
references.pmid: 16934839
references.citation: J Bacteriol. 1999 Sep;181(17):5395-401
references.pmid: 10464212
references.citation: Mol Microbiol. 2006 Dec;62(6):1655-73
references.pmid: 17427288
references.citation: BMC Microbiol. 2010 Dec 29;10:327
references.pmid: 21190553
references.citation: Mol Microbiol. 2007 Aug;65(3):684-99
references.pmid: 17635188
references.citation: J Biol Chem. 1988 Jul 25;263(21):10088-95
references.pmid: 3292521
references.citation: J Biol Chem. 2005 Jan 7;280(1):326-33
references.pmid: 15522883
references.citation: J Bacteriol. 2005 Apr;187(7):2540-50
references.pmid: 15774900
references.citation: Microbiology (Reading). 2006 Feb;152(Pt 2):493-504
references.pmid: 16436437
references.citation: Clin Microbiol Rev. 2011 Jan;24(1):71-109
references.pmid: 21233508
references.citation: Eur J Biochem. 1995 May 15;230(1):80-7
references.pmid: 7601127
references.citation: Nature. 1999 Aug 12;400(6745):693-6
references.pmid: 10458167
references.citation: Annu Rev Biochem. 1995;64:29-63
references.pmid: 7574484
references.citation: Biochim Biophys Acta. 2008 Jan;1784(1):193-202
references.pmid: 17869195
references.citation: Genes Dev. 2005 Jul 15;19(14):1692-704
references.pmid: 15985609
references.citation: J Mol Biol. 2009 Feb 20;386(2):451-64
references.pmid: 19121323
references.citation: Microbiology (Reading). 2008 Sep;154(Pt 9):2796-2803
references.pmid: 18757813
references.citation: J Bacteriol. 1982 Sep;151(3):1109-17
references.pmid: 6125497
references.citation: Biochem J. 2009 Jun 26;421(2):263-72
references.pmid: 19400768
references.citation: J Antimicrob Chemother. 2011 Aug;66(8):1766-72
references.pmid: 21622974
references.citation: Biochimie. 2010 Dec;92(12):1793-800
references.pmid: 20659527
references.citation: Protein Cell. 2010 Nov;1(11):1011-22
references.pmid: 21153518
references.citation: Microbiol Mol Biol Rev. 2008 Mar;72(1):126-56, table of contents
references.pmid: 18322037
references.citation: Tuber Lung Dis. 1997;78(5-6):211-23
references.pmid: 10209675
references.citation: J Biochem. 1973 Sep;74(3):525-38
references.pmid: 4756894
references.citation: J Biol Chem. 2006 Oct 6;281(40):30094-103
references.pmid: 16873379
references.citation: Microbiol Mol Biol Rev. 2005 Dec;69(4):585-607
references.pmid: 16339737
references.citation: Protein Expr Purif. 2006 Jan;45(1):175-82
references.pmid: 16168669
references.citation: Proc Natl Acad Sci U S A. 2010 Apr 20;107(16):7521-6
references.pmid: 20368441
references.citation: Nat Prod Rep. 2001 Oct;18(5):503-19
references.pmid: 11699883
references.citation: Proc Natl Acad Sci U S A. 2006 Jul 25;103(30):11346-51
references.pmid: 16844784
references.citation: Protein Sci. 1996 Dec;5(12):2566-74
references.pmid: 8976565
references.citation: J Antimicrob Chemother. 2010 Oct;65(10):2101-7
references.pmid: 20719764
references.citation: J Biol Chem. 2006 Dec 29;281(52):40107-13
references.pmid: 17068335
references.citation: J Biol Chem. 2008 Dec 26;283(52):36553-63
references.pmid: 18974047
references.citation: J Bacteriol. 2000 Dec;182(23):6827-30
references.pmid: 11073931
references.citation: Nature. 2011 Jan 27;469(7331):483-90
references.pmid: 21270886
references.citation: J Bacteriol. 2006 Jan;188(1):19-27
references.pmid: 16352817
references.citation: J Biol Chem. 1968 May 25;243(10):2665-72
references.pmid: 4967958
references.citation: Glycobiology. 2001 Sep;11(9):107R-118R
references.pmid: 11555614
references.citation: Mol Microbiol. 2006 Feb;59(3):989-99
references.pmid: 16420366
references.citation: Trends Microbiol. 2000 May;8(5):238-44
references.pmid: 10785641
references.citation: Microbiology (Reading). 1997 Mar;143 ( Pt 3):921-928
references.pmid: 9084176
references.citation: Comput Biol Chem. 2005 Oct;29(5):368-78
references.pmid: 16213791
references.citation: Int J Antimicrob Agents. 2012 Feb;39(2):124-9
references.pmid: 22079533
references.citation: J Biol Chem. 2006 Jun 9;281(23):15680-6
references.pmid: 16595662
references.citation: Biochem J. 2009 Mar 1;418(2):369-78
references.pmid: 19014350
references.citation: Chem Biol. 2010 Aug 27;17(8):820-30
references.pmid: 20797611
references.citation: Biochemistry. 1990 Mar 6;29(9):2379-86
references.pmid: 2186811
references.citation: J Bacteriol. 2003 Nov;185(22):6507-12
references.pmid: 14594822
references.citation: J Bacteriol. 2005 Apr;187(8):2747-57
references.pmid: 15805521
references.citation: Biochem J. 2008 Oct 1;415(1):27-33
references.pmid: 18557704
references.citation: Microbiol Mol Biol Rev. 1998 Dec;62(4):1079-93
references.pmid: 9841666
references.citation: Antimicrob Agents Chemother. 2003 Jan;47(1):283-91
references.pmid: 12499203
references.citation: Mol Microbiol. 2010 Mar;75(5):1064-77
references.pmid: 20487298
references.citation: Gene. 1993 Jan 15;123(1):17-24
references.pmid: 8380783
references.citation: Eur J Biochem. 1991 Dec 18;202(3):1169-76
references.pmid: 1765076
references.citation: Tuberculosis (Edinb). 2010 Jan;90(1):16-24
references.pmid: 19945347
references.citation: Mol Microbiol. 2010 May;76(3):616-33
references.pmid: 20233306
references.citation: FEMS Microbiol Lett. 2006 Sep;262(1):39-47
references.pmid: 16907737
references.citation: J Mol Biol. 1999 Jun 11;289(3):579-90
references.pmid: 10356330