Biochemical and Structural Analysis of a Novel Esterase from Caulobacter crescentus related to Penicillin-Binding Protein (PBP)

Abstract

Considering that the prevalence of antibiotic-resistant pathogenic bacteria is largely increasing, a thorough understanding of penicillin-binding proteins (PBPs) is of great importance and crucial significance because this enzyme family is a main target of β-lactam-based antibiotics. In this work, combining biochemical and structural analysis, we present new findings that provide novel insights into PBPs. Here, a novel PBP homologue (CcEstA) from Caulobacter crescentus CB15 was characterized using native-PAGE, mass spectrometry, gel filtration, CD spectroscopy, fluorescence, reaction kinetics, and enzyme assays toward various substrates including nitrocefin. Furthermore, the crystal structure of CcEstA was determined at a 1.9 Å resolution. Structural analyses showed that CcEstA has two domains: a large α/β domain and a small α-helix domain. A nucleophilic serine (Ser68) residue is located in a hydrophobic groove between the two domains along with other catalytic residues (Lys71 and Try157). Two large flexible loops (UL and LL) of CcEstA are proposed to be involved in the binding of incoming substrates. In conclusion, CcEstA could be described as a paralog of the group that contains PBPs and β-lactamases. Therefore, this study could provide new structural and functional insights into the understanding this protein family.

Molecular Characterization of a Novel Family VIII Esterase with β-Lactamase Activity (PsEstA) from Paenibacillus sp.

“…In sequence analysis, PsEstA was found to contain a higher percentage of acidic (Asp and Glu, 12.0%) than basic (Lys and Arg, 9.3%) amino acids. The structural model of PsEstA consisted of a large α/β domain and a small α-helical domain, and these features are frequently observed in other β-lactamases and family VIII esterases [14,17,34,35]. The nucleophilic serine was found to be located near the surface of the tunnel formed The structural model of PsEstA consisted of a large α/β domain and a small α-helical domain, and these features are frequently observed in other β-lactamases and family VIII esterases [14,17,34,35].…”

“…For the overlay activity assay, native-PAGE analysis was performed. Subsequently, the gel was washed three times with a storage buffer, and then incubated with 4-methylumbelliferyl (4-MU) acetate to detect the fluorescent signal under UV illumination [16,17]. Hydrolysis of 4-methylumbelliferone (4-MU) acetate or phosphate by PsEstA was also investigated in an Eppendorf tube in an ultraviolet (UV) illumination box.…”

“…The β-lactamase activity of PsEstA was determined using the chromogenic β-lactam substrate nitrocefin [17]. PsEstA (0.1 mg/mL) was reacted with a 484 µM nitrocefin solution in 20 mM Tris-HCl pH 8.5 at 25 • C, and the color change in the reaction mixture was observed.…”

“…Interestingly, several esterases, collectively known as family VIII esterases, have recently been reported to have similarities with class C β-lactamases and hydrolyze nitrocefin or other β-lactam antibiotics [12][13][14][15]. These proteins have a Ser-Lys-Tyr catalytic triad, and their nucleophilic serine residue is present in the S-X-X-K motif located in the N-terminal region, instead of the G-X-S-X-G motif in most bacterial lipases/esterases [16,17]. A novel family VIII esterase with β-lactamase activity (PsEstA) has recently been identified in Paenibacillus sp.…”

Identification and Crystallization of Penicillin-Binding Protein/β-Lactamase Homolog (Rp46) from Ruegeria Pomeroyi

“…To date, a very limited number of crystal structures of PBP-βL homologs is known including an esterase (EstB) from Burkholderia gladioli [20], a simvastatin synthase (LovD) from Aspergillus terreus [21], a family VIII carboxylesterase (EstU1) [22], Est-Y29 from a metagenomic library [23], and a PBP-like esterase (CcEstA) [24]. Furthermore, the catalytic mechanisms and physiological functions of this enzyme family remain mainly unclear.…”