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Structural and functional analysis of de‐ N ‐acetylase PgaB from periodontopathogen Aggregatibacter actinomycetemcomitans
Author(s) -
Parthiban C.,
Varudharasu D.,
Shanmugam M.,
Gopal P.,
Ragunath C.,
Thomas L.,
Nitz M.,
Ramasubbu N.
Publication year - 2017
Publication title -
molecular oral microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.18
H-Index - 77
eISSN - 2041-1014
pISSN - 2041-1006
DOI - 10.1111/omi.12175
Subject(s) - active site , enzyme , operon , biochemistry , aggregatibacter actinomycetemcomitans , bacillus subtilis , chemistry , stereochemistry , mutant , biology , bacteria , gene , genetics , porphyromonas gingivalis
Summary 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 ( Aa PgaB) and the N‐terminal catalytic domain (residues 25–290, Aa Pga BN ) from A. actinomycetemcomitans were cloned, expressed and purified. The enzymatic activities of the Aa PgaB enzymes were determined using 7‐acetoxycoumarin‐3‐carboxylic acid as the substrate. The Aa PgaB enzymes displayed significantly lower de‐ N ‐acetylase activity compared with the activity of the deacetylase PdaA from Bacillus subtilis , a member of the CE 4 family of enzymes. To delineate the differences in the activity and the active site architecture, the structure of Aa Pga BN was determined. The Aa Pga BN structure has two metal ions in the active site instead of one found in other CE 4 enzymes. Based on the crystal structure comparisons among the various CE 4 enzymes, two residues, Q51 and R271, were identified in Aa PgaB, which could potentially affect the enzyme activity. Of the two mutants generated, Q51E and R271K, the variant Q51E showed enhanced activity compared with Aa PgaB, 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 CE 4 family of enzymes, evidence that Aa Pga BN is catalytically active and that mutant Q51E exhibits higher de‐ N ‐acetylase activity.