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.

Role of de‐N‐acetylase PgaB from Aggregatibacter actinomycetemcomitans in exopolysaccharide export in biofilm mode of growth

“…The PNAG deacetylase enzyme PgaB of A. actinomycetemcomitans has been characterized previously in our laboratory and we showed that the N‐terminal catalytic domain of PgaB is responsible for the deacetylase activity . The results reported here indicate that in the absence of the catalytic domain of PgaB, the pga operon transcription ( pgaA , pgaB and pgaC ) is downregulated, which leads to less PNAG production.…”

“…The protein sequence of Aa PgaB is very similar to that of Ec PgaB and the structures are highly superimposable . In E. coli , the non‐polar ΔpgaB strain was shown to be unable to export PNAG although they were able to produce PNAG .…”

“…Among these four proteins, PgaB of both E. coli ( Ec PgaB) and A. actinomycetemcomitans ( Aa PgaB), and IcaB of S. epidermidis ( Se IcaB), possess a de‐ N ‐acetylase domain, which removes the N ‐acetyl groups from GlcNAc residues that make up the exopolysaccharide . The proteins PgaB/IcaB are interesting because they confer positive charges to PNAG to promote PNAG association with the cell surface lipopolysaccharides in A. actinomycetemcomitans or in S. aureus …”

Substrate Recognition and Specificity of Chitin Deacetylases and Related Family 4 Carbohydrate Esterases

“…Other poly-β-1,6-GlcNAc de- N -acetylases with solved X-ray structures, so far, are Ad IcaB from Ammoniex degensii [ 77 ], Bb BpsB from Bordetella bronchiseptica [ 45 ], and Aa PgaB from Aggregatibacter actinomycetemcomitans [ 147 ]. All of these enzymes show low activity on β-1,6-glucan, a common property of this enzyme subclass within family CE4.…”

“…Acetylxylan esterases from S. lividans and C. thermocelum [ 74 ] show the canonical active topology and metal coordination in this enzyme family, as well as other peptidoglycan deacetylases ( S. mutants [ 74 ], B. anthracis [ 69 ], and B. cereus [ 150 ]). The case of poly-β-1,6-GlcNAc deacetylases is different [ 75 , 76 , 77 , 147 ], as they have a circularly permutated CE4 domain and some variations in the positioning of active site residues, as discussed below. It was not until 2014 that the first crystal structure of a CE4 enzyme in complex with substrates was solved, that of the Vibrio cholera CDA [ 62 ], providing new information on loop organization and insights into the structural determinants of substrate binding, specificity, and catalysis.…”