title: Structural and functional analysis of de-N-acetylase PgaB from periodontopathogen Aggregatibacter actinomycetemcomitans.
abstract: The oral pathogen Aggregatibacter actinomycetemcomitans uses pga gene locus for the production of an exopolysaccharide made up of a linear homopolymer of β-1,6-N-acetyl-d-glucosamine (PGA). An enzyme encoded by the pgaB of the pga operon in A. actinomycetemcomitans is a de-N-acetylase, which is used to alter the PGA. The full length enzyme (AaPgaB) and the N-terminal catalytic domain (residues 25-290, AaPgaBN) from A. actinomycetemcomitans were cloned, expressed and purified. The enzymatic activities of the AaPgaB enzymes were determined using 7-acetoxycoumarin-3-carboxylic acid as the substrate. The AaPgaB enzymes displayed significantly lower de-N-acetylase activity compared with the activity of the deacetylase PdaA from Bacillus subtilis, a member of the CE4 family of enzymes. To delineate the differences in the activity and the active site architecture, the structure of AaPgaBN was determined. The AaPgaBN structure has two metal ions in the active site instead of one found in other CE4 enzymes. Based on the crystal structure comparisons among the various CE4 enzymes, two residues, Q51 and R271, were identified in AaPgaB, which could potentially affect the enzyme activity. Of the two mutants generated, Q51E and R271K, the variant Q51E showed enhanced activity compared with AaPgaB, validating the requirement that an activating aspartate residue in the active site is essential for higher activity. In summary, our study provides the first structural evidence for a di-nuclear metal site at the active site of a member of the CE4 family of enzymes, evidence that AaPgaBN is catalytically active and that mutant Q51E exhibits higher de-N-acetylase activity.
authors.affiliation: Department of Oral Biology, Rutgers School of Dental Medicine, Newark, NJ, USA.
authors.forename: C
authors.initials: C
authors.lastname: Parthiban
authors.affiliation: Selvam Structure Based Drug Design Laboratory, Selvam College of Technology, Namakkal, Tamilnadu, India.
authors.forename: D
authors.initials: D
authors.lastname: Varudharasu
authors.affiliation: Department of Oral Biology, Rutgers School of Dental Medicine, Newark, NJ, USA.
authors.forename: M
authors.initials: M
authors.lastname: Shanmugam
authors.affiliation: Department of Oral Biology, Rutgers School of Dental Medicine, Newark, NJ, USA.
authors.forename: P
authors.initials: P
authors.lastname: Gopal
authors.affiliation: Scientific Chemical Technologies, Malden, MA, USA.
authors.forename: C
authors.initials: C
authors.lastname: Ragunath
authors.affiliation: Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK, USA.
authors.forename: L
authors.initials: L
authors.lastname: Thomas
authors.affiliation: Department of Chemistry, University of Toronto, Toronto, ON, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Nitz
authors.affiliation: Department of Oral Biology, Rutgers School of Dental Medicine, Newark, NJ, USA.
authors.forename: N
authors.initials: N
authors.lastname: Ramasubbu
mesh_terms: D000115:Acetylesterase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016976:Aggregatibacter actinomycetemcomitans / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D001426:Bacterial Proteins / Q000235:genetics; D018441:Biofilms / Q000254:growth & development; D020134:Catalytic Domain; D003460:Crystallization; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D009876:Operon; D011135:Polysaccharides, Bacterial; D000072417:Protein Domains; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008670:Metals; D011135:Polysaccharides, Bacterial; D000115:Acetylesterase
keywords: biofilm; deacetylation; exopolysaccharide; kinetics; oral pathogen