title: Structural and biochemical analyses of Mycobacterium tuberculosis N-acetylmuramyl-L-alanine amidase Rv3717 point to a role in peptidoglycan fragment recycling.
abstract: Peptidoglycan hydrolases are key enzymes in bacterial cell wall homeostasis. Understanding the substrate specificity and biochemical activity of peptidoglycan hydrolases in Mycobacterium tuberculosis is of special interest as it can aid in the development of new cell wall targeting therapeutics. In this study, we report biochemical and structural characterization of the mycobacterial N-acetylmuramyl-L-alanine amidase, Rv3717. The crystal structure of Rv3717 in complex with a dipeptide product shows that, compared with previously characterized peptidoglycan amidases, the enzyme contains an extra disulfide-bonded β-hairpin adjacent to the active site. The structure of two intermediates in assembly reveal that Zn(2+) binding rearranges active site residues, and disulfide formation promotes folding of the β-hairpin. Although Zn(2+) is required for hydrolysis of muramyl dipeptide, disulfide oxidation is not required for activity on this substrate. The orientation of the product in the active site suggests a role for a conserved glutamate (Glu-200) in catalysis; mutation of this residue abolishes activity. The product binds at the head of a closed tunnel, and the enzyme showed no activity on polymerized peptidoglycan. These results point to a potential role for Rv3717 in peptidoglycan fragment recycling.
authors.affiliation: From the Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3220.
authors.forename: Daniil M
authors.initials: DM
authors.lastname: Prigozhin
authors.forename: Daniela
authors.initials: D
authors.lastname: Mavrici
authors.forename: John P
authors.initials: JP
authors.lastname: Huizar
authors.forename: Hilary J
authors.initials: HJ
authors.lastname: Vansell
authors.forename: Tom
authors.initials: T
authors.lastname: Alber
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004151:Dipeptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D010457:Peptidoglycan / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004151:Dipeptides; D004220:Disulfides; D010457:Peptidoglycan; D000581:Amidohydrolases; C019665:amidase; D015032:Zinc
keywords: Cell Wall; Crystal Structure; Hydrolases; Mycobacterium tuberculosis; Peptidoglycan
references.citation: Microbiology (Reading). 2000 Feb;146 ( Pt 2):249-262
references.pmid: 10708363
references.citation: J Biol Chem. 1966 Aug 10;241(15):3462-7
references.pmid: 4958491
references.citation: J Mol Biol. 2006 Dec 8;364(4):678-89
references.pmid: 17010991
references.citation: Microbiol Mol Biol Rev. 2008 Mar;72(1):126-56, table of contents
references.pmid: 18322037
references.citation: J Mol Biol. 2011 Oct 14;413(1):247-60
references.pmid: 21864539
references.citation: Science. 2010 May 14;328(5980):852-6
references.pmid: 20466922
references.citation: Microbiol Mol Biol Rev. 2011 Dec;75(4):636-63
references.pmid: 22126997
references.citation: Nat Rev Immunol. 2011 Nov 11;11(12):837-51
references.pmid: 22076558
references.citation: Glycobiology. 2001 Sep;11(9):107R-118R
references.pmid: 11555614
references.citation: Microbiol Mol Biol Rev. 2008 Jun;72(2):211-27, table of contents
references.pmid: 18535144
references.citation: J Synchrotron Radiat. 2004 Nov 1;11(Pt 6):447-55
references.pmid: 15496731
references.citation: FEMS Immunol Med Microbiol. 2010 Feb;58(1):39-50
references.pmid: 19799629
references.citation: J Gen Microbiol. 1984 Sep;130(9):2395-402
references.pmid: 6150066
references.citation: Biochim Biophys Acta. 2005 Feb 14;1747(1):57-66
references.pmid: 15680239
references.citation: Protein Expr Purif. 2005 May;41(1):207-34
references.pmid: 15915565
references.citation: J Comput Chem. 2004 Oct;25(13):1605-12
references.pmid: 15264254
references.citation: Nucleic Acids Res. 2010 Jan;38(Database issue):D211-22
references.pmid: 19920124
references.citation: Mol Microbiol. 2012 Aug;85(4):768-81
references.pmid: 22715947
references.citation: Appl Microbiol Biotechnol. 2011 Oct;92(1):1-11
references.pmid: 21796380
references.citation: J Mol Biol. 2004 Jul 16;340(4):783-95
references.pmid: 15223320
references.citation: Methods Mol Biol. 2008;426:419-35
references.pmid: 18542881
references.citation: J Bacteriol. 2011 Oct;193(19):5477-86
references.pmid: 21803993
references.citation: Trends Microbiol. 2012 Nov;20(11):540-7
references.pmid: 22944244
references.citation: PLoS Comput Biol. 2011 Oct;7(10):e1002195
references.pmid: 22039361
references.citation: PLoS Pathog. 2011 Sep;7(9):e1002251
references.pmid: 21980284
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
references.pmid: 15572765