Structural and biochemical analysis of penicillin-binding protein 2 from Campylobacter jejuni.

Penicillin-binding protein 2 (PBP2) plays a key role in the formation of peptidoglycans in bacterial cell walls by crosslinking glycan chains through transpeptidase activity. PBP2 is also found in Campylobacter jejuni, a pathogenic bacterium that causes food-borne enteritis in humans. To elucidate the essential structural features of C. jejuni PBP2 (cjPBP2) that mediate its biological function, we determined the crystal structure of cjPBP2 and assessed its protein stability under various conditions. cjPBP2 adopts an elongated two-domain structure, consisting of a transpeptidase domain and a pedestal domain, and contains typical active site residues necessary for transpeptidase activity, as observed in other PBP2 proteins. Moreover, cjPBP2 responds to ß-lactam antibiotics, including ampicillin, cefaclor, and cefmetazole, suggesting that ß-lactam antibiotics inactivate cjPBP2. In contrast to typical PBP2 proteins, cjPBP2 is a rare example of a Zn2+-binding PBP2 protein, as the terminal structure of its transpeptidase domain accommodates a Zn2+ ion via three cysteine residues and one histidine residue. Zn2+ binding helps improve the protein stability of cjPBP2, providing opportunities to develop new C. jejuni-specific antibacterial drugs that counteract the Zn2+-binding ability of cjPBP2.

Structural and biochemical analysis of the GDSL-family esterase CJ0610C from Campylobacter jejuni.

GDSL domain-containing proteins generally hydrolyze esters or lipids and play critical roles in diverse biological and industrial processes. GDSL hydrolases use catalytic triad and oxyanion hole residues from conserved blocks I, II, III, and V to drive the esterase reaction. However, GDSL hydrolases exhibit large deviations in sequence, structure, and substrate specificity, requiring the characterization of each GDSL hydrolase to reveal its catalytic mechanism. We identified a GDSL protein (CJ0610C) from pathogenic Campylobacter jejuni and assessed its biochemical and structural features. CJ0610C displayed esterase activity for p-nitrophenyl acetate and preferred short chain esters and alkaline pH. The C-terminal two-thirds of CJ0610C corresponding to the GDSL domain forms a three-layered α/ß/α fold as a core structure in which a five-stranded ß-sheet is sandwiched by α-helices. In the CJ0610C structure, conserved catalytic triad and oxyanion hole residues that are indispensable for esterase activity are found in blocks I, III, and V. However, CJ0610C lacks the conserved block-II glycine residue and instead employs a unique asparagine residue as another oxyanion hole residue. Moreover, our structural analysis suggests that substrate binding is mediated by a CJ0610C-specific pocket, which is surrounded by hydrophobic residues and occluded at one end by a positively charged arginine residue.

Structural analysis of the pseudaminic acid synthase PseI from Campylobacter jejuni.

Campylobacter jejuni PseI is a pseudaminic acid synthase that condenses the 2,4-diacetamido-2,4,6-trideoxy-l-altrose sugar (6-deoxy AltdiNAc) and phosphoenolpyruvate to generate pseudaminic acid, a sialic acid-like 9-carbon backbone α-keto sugar. Pseudaminic acid is conjugated to cell surface proteins and lipids and plays a key role in the mobility and virulence of C. jejuni and other pathogenic bacteria. To provide insights into the catalytic mechanism of PseI, we performed a structural study on PseI. PseI forms a two-domain structure and assembles into a domain-swapped homodimer. The PseI dimer has two cavities, each of which accommodates a metal ion using conserved histidine residues. A comparative analysis of structures and sequences suggests that the cavity of PseI functions as an active site that binds the 6-deoxy AltdiNAc and phosphoenolpyruvate substrates and mediates their condensation. Furthermore, we propose the substrate binding-induced structural rearrangement of PseI and predict 6-deoxy AltdiNAc recognition residues that are specific to PseI.