Structure and metal-dependent mechanism of peptidoglycan deacetylase, a streptococcal virulence factor

Abstract

Streptococcus pneumoniae peptidoglycan GlcNAc deacetylase (SpPgdA) protects the Gram-positive bacterial cell wall from host lysozymes by deacetylating peptidoglycan GlcNAc residues. Deletion of the pgda gene has been shown to result in hypersensitivity to lysozyme and reduction of infectivity in a mouse model. SpPgdA is a member of the family 4 carbohydrate esterases, for which little structural information exists, and no catalytic mechanism has yet been defined. Here we describe the native crystal structure and product complexes of SpPgdA biochemical characterization and mutagenesis. The structural data show that SpPgdA is an elongated three-domain protein in the crystal. The structure, in combination with mutagenesis, shows that SpPgdA is a metalloenzyme using a His-His-Asp zinc-binding triad with a nearby aspartic acid and histidine acting as the catalytic base and acid, respectively, somewhat similar to other zinc deacetylases such as LpxC. The enzyme is able to accept GlcNAc 3 as a substrate (Km ‫؍‬ 3.8 mM, kcat ‫؍‬ 0.55 s ؊1 ), with the N-acetyl of the middle sugar being removed by the enzyme. The data described here show that SpPgdA and the other family 4 carbohydrate esterases are metalloenzymes and present a step toward identification of mechanismbased inhibitors for this important class of enzymes.crystal structure ͉ metalloenzyme

Structure and Mechanism of Chitin Deacetylase from the Fungal Pathogen Colletotrichum lindemuthianum,

“…The C-terminal hexa-His-tagged ClCDA protein expressed in P. pastoris was assayed using the fluorogenic labeling method previously described (34). Standard reactions consisted of 100 nM protein (dialyzed into doubly distilled H 2 O), 50 mM Bis-Tris, pH 7, and 0.6 mM (GlcNAc) 3 in a total volume of 50 µL, incubated for 10 min at 37°C.…”

Understanding the structural and functional properties of carbohydrate esterases with a special focus on hemicellulose deacetylating acetyl xylan esterases

“…Unlike other class-1, 2 and 3 CE class enzymes, CE class-4 enzymes are highly specific towards the acetyl xylan substrates and these AcXEs are not active against acetylgalactoglucomannan or acetylated manno-residues. However, the classification of chitin and peptidoglycan deacetylating AcXEs under CE class-4 explains the characteristic ability of CE class-4 AcXEs activity on chitin (Biely et al 1996b; Caufrier et al 2003; Blair et al 2005; Taylor et al 2006). The CE class-5 AcXEs show their specificity towards acetyl xylan residues, acetylated xylo-oligosaccharide, mannosides and cellulose acetate residues by deacetylating them on position 2.…”

Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases

“…CE4 AcXEs operate an acid (aspartic acid) -base (histidine) catalytic mechanism and are also known as 'NodB homologs' [69][70][71]. They possess the highly conserved catalytic NodB domain [72] characteristic of other rhizobial NodB enzymes in the CE4 family such as the chitin deacetylases, chitooligosaccharide deacetylases, rhizobial nod factor deacetylases as well as peptidoglycan N-acetylglucosamine deacetylases and Nacetylmuramic deacetylases which de-esterify N-or O-acetyl bonds of plant cell wall polymers or oligomers [73][74][75]. Family CE4 AcXEs possess an eight-stranded (β-α)8 barrel protein fold which has an irregular structure [38].…”

“…Family CE4 AcXEs possess an eight-stranded (β-α)8 barrel protein fold which has an irregular structure [38]. CE4 AcXEs prefer longer chain oligosaccharide substrates and exhibit positional deacetylation specificity [67,73]. They are active on various acetylated xylan residues and are reported to show preference for methyl per-O-acetyl-β-Dxylopyranoside [25], but unlike most AcXEs in other families, do not show activity on paranitrophenyl acetate (pNPA) or 4-methylumbelliferyl acetate [67,73].…”

“…CE4 AcXEs prefer longer chain oligosaccharide substrates and exhibit positional deacetylation specificity [67,73]. They are active on various acetylated xylan residues and are reported to show preference for methyl per-O-acetyl-β-Dxylopyranoside [25], but unlike most AcXEs in other families, do not show activity on paranitrophenyl acetate (pNPA) or 4-methylumbelliferyl acetate [67,73]. On the contrary, an enzyme recently reported as a CE4 AcXE from Anoxybacillus flavithermus showed activity on pNPA, but its activity on acetylated xylan was not tested to verify its identity as a 'true' AcXE [76].…”