Title
Biochemical and Structural Analysis of a Novel Esterase from Caulobacter crescentus related to Penicillin-Binding Protein (PBP)
Author
Bum Han Ryu, Tri Duc Ngo, Wanki Yoo, Sojeong Lee, Boo-Young Kim, Euijoo Lee, Kyeong Kyu Kim, T. Doohun Kim
Year
2016
Journal
Scientific Reports
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.
Full Article
Instrument
FP-6200
Keywords
Fluorescence, Protein structure, Chemical stability, Protein denaturation, Biochemistry