title: Mycobacteriophage Ms6 LysA: a peptidoglycan amidase and a useful analytical tool.
abstract: Since the peptidoglycan isolated from Mycobacterium spp. is refractory to commercially available murolytic enzymes, possibly due to the presence of various modifications found on this peptidoglycan, the utility of a mycobacteriophage-derived murolytic enzyme was assessed for an analysis of peptidoglycan from mycobacteria. We cloned, expressed, and purified the lysA gene product, a protein with homology to known peptidoglycan-degrading amidases, from bacteriophage Ms6. The recombinant protein was shown to cleave the bond between l-Ala and d-muramic acid of muramyl pentapeptide and to release up to 70% of the diaminopimelic acid present in the isolated mycobacterial cell wall. In contrast to lysozyme, which, in culture, inhibits the growth of both Mycobacterium smegmatis and Mycobacterium tuberculosis, LysA had no effect on the growth of either species. However, the enzyme is useful for solubilizing the peptide chains of isolated mycobacterial peptidoglycan for analysis. The data indicate that the stem peptides from M. smegmatis are heavily amidated, containing few free carboxylic acids, regardless of the cross-linking status.
authors.affiliation: Department of Microbiology Immunology and Pathology, Colorado State University, Fort Collins, CO, USA.
authors.forename: Sebabrata
authors.initials: S
authors.lastname: Mahapatra
authors.forename: Charles
authors.initials: C
authors.lastname: Piechota
authors.forename: Filipa
authors.initials: F
authors.lastname: Gil
authors.forename: Yufang
authors.initials: Y
authors.lastname: Ma
authors.forename: Hairong
authors.initials: H
authors.lastname: Huang
authors.forename: Michael S
authors.initials: MS
authors.lastname: Scherman
authors.forename: Victoria
authors.initials: V
authors.lastname: Jones
authors.forename: Martin S
authors.initials: MS
authors.lastname: Pavelka
authors.forename: Jose
authors.initials: J
authors.lastname: Moniz-Pereira
authors.forename: Madalena
authors.initials: M
authors.lastname: Pimentel
authors.forename: Michael R
authors.initials: MR
authors.lastname: McNeil
authors.forename: Dean C
authors.initials: DC
authors.lastname: Crick
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism*; D002473:Cell Wall; D003001:Cloning, Molecular; D003960:Diaminopimelic Acid / Q000378:metabolism; D015870:Gene Expression; D009160:Mycobacteriophages / Q000201:enzymology* / Q000235:genetics; D009161:Mycobacterium / Q000187:drug effects*; D010457:Peptidoglycan / Q000378:metabolism*
chemical_list: D010457:Peptidoglycan; D003960:Diaminopimelic Acid; D000581:Amidohydrolases; C019665:amidase