Crystallographic analysis and biochemical applications of a novel penicillin-binding protein/β-lactamase homologue from a metagenomic library

Interest in penicillin-binding proteins and β-lactamases (the PBP-βL family) is increasing owing to their biological and clinical significance. In this study, the crystal structure of Est-Y29, a metagenomic homologue of the PBP-βL family, was determined at 1.7 Å resolution. In addition, complex structures of Est-Y29 with 4-nitrophenyl phosphate (4NP) and with diethyl phosphonate (DEP) at 2.0 Å resolution were also elucidated. Structural analyses showed that Est-Y29 is composed of two domains: a β-lactamase fold and an insertion domain. A deep hydrophobic patch between these domains defines a wide active site, and a nucleophilic serine (Ser58) residue is located in a groove defined primarily by hydrophobic residues between the two domains. In addition, three hydrophobic motifs, which make up the substrate-binding site, allow this enzyme to hydrolyze a wide variety of hydrophobic compounds, including fish and olive oils. Furthermore, cross-linked Est-Y29 aggregates (CLEA-Est-Y29) significantly increase the stability of the enzyme as well as its potential for extensive reuse in various deactivating conditions. The structural features of Est-Y29, together with biochemical and biophysical studies, could provide a molecular basis for understanding the properties and regulatory mechanisms of the PBP-βL family and their potential for use in industrial biocatalysts.

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PBP5 Complementation of a PBP3 Deficiency in Enterococcus hirae

Journal of Bacteriology ◽

10.1128/jb.00334-06 ◽

2006 ◽

Vol 188 (17) ◽

pp. 6298-6307 ◽

Cited By ~ 4

Author(s):

S. Leimanis ◽

N. Hoyez ◽

S. Hubert ◽

M. Laschet ◽

Eric Sauvage ◽

...

Keyword(s):

Site Directed Mutagenesis ◽

Penicillin Binding Protein ◽

Enterococcus Hirae ◽

Penicillin Binding Proteins ◽

Protein Protein Interaction ◽

Interaction Sites ◽

Functional Potential ◽

The Stability ◽

Protein Interaction Sites ◽

Made In

ABSTRACT The low susceptibility of enterococci to β-lactams is due to the activity of the low-affinity penicillin-binding protein 5 (PBP5). One important feature of PBP5 is its ability to substitute for most, if not all, penicillin-binding proteins when they are inhibited. That substitution activity was analyzed in Enterococcus hirae SL2, a mutant whose pbp5 gene was interrupted by the nisRK genes and whose PBP3 synthesis was submitted to nisin induction. Noninduced SL2 cells were unable to divide except when plasmid-borne pbp5 genes were present, provided that the PBP5 active site was functional. Potential protein-protein interaction sites of the PBP5 N-terminal module were mutagenized by site-directed mutagenesis. The T167-L184 region (designated site D) appeared to be an essential intramolecular site needed for the stability of the protein. Mutations made in the two globular domains present in the N-terminal module indicated that they were needed for the suppletive activity. The P197-N209 segment (site E) in one of these domains seemed to be particularly important, as single and double mutations reduced or almost completely abolished, respectively, the action of PBP5.

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Cryo-EM structure of amyloid fibrils formed by the entire low complexity domain of TDP-43

Nature Communications ◽

10.1038/s41467-021-21912-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Qiuye Li ◽

W. Michael Babinchak ◽

Witold K. Surewicz

Keyword(s):

Amyloid Fibrils ◽

Low Complexity ◽

Structural Features ◽

Protein Fragments ◽

Hydrophobic Residues ◽

Backbone Conformation ◽

Hydrophobic Amino Acids ◽

Insight Into ◽

Lateral Sclerosis

AbstractAmyotrophic lateral sclerosis and several other neurodegenerative diseases are associated with brain deposits of amyloid-like aggregates formed by the C-terminal fragments of TDP-43 that contain the low complexity domain of the protein. Here, we report the cryo-EM structure of amyloid formed from the entire TDP-43 low complexity domain in vitro at pH 4. This structure reveals single protofilament fibrils containing a large (139-residue), tightly packed core. While the C-terminal part of this core region is largely planar and characterized by a small proportion of hydrophobic amino acids, the N-terminal region contains numerous hydrophobic residues and has a non-planar backbone conformation, resulting in rugged surfaces of fibril ends. The structural features found in these fibrils differ from those previously found for fibrils generated from short protein fragments. The present atomic model for TDP-43 LCD fibrils provides insight into potential structural perturbations caused by phosphorylation and disease-related mutations.

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Increasing Ceftriaxone Resistance and Multiple Alterations of Penicillin-Binding Proteins among Penicillin-Resistant Streptococcus pneumoniae Isolates in Taiwan

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01563-06 ◽

2007 ◽

Vol 51 (9) ◽

pp. 3404-3406 ◽

Cited By ~ 20

Author(s):

Cheng-Hsun Chiu ◽

Lin-Hui Su ◽

Yhu-Chering Huang ◽

Jui-Chia Lai ◽

Hsiu-Ling Chen ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Amino Acid ◽

Binding Proteins ◽

Binding Protein ◽

Penicillin Binding Protein ◽

Penicillin Binding Proteins

ABSTRACT The rate of nonsusceptibility of penicillin-resistant Streptococcus pneumoniae strains to ceftriaxone increased significantly in Taiwan in 2005. Approximately 90% of the ceftriaxone-nonsusceptible isolates were found to be of four major serotypes (serotypes 6B, 14, 19F, and 23F). Seven amino acid alterations in the penicillin-binding protein 2B transpeptidase-encoding region specifically contributed to the resistance.

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FABRICATION OF NANOPOROUS CHITOSAN MEMBRANES

NANO ◽

10.1142/s1793292010001846 ◽

2010 ◽

Vol 05 (01) ◽

pp. 53-60 ◽

Cited By ~ 1

Author(s):

XIAOLIANG WANG ◽

XIANG LI ◽

ELEANOR STRIDE ◽

MOHAN EDIRISINGHE

Keyword(s):

Low Frequency ◽

Drug Carriers ◽

Structural Features ◽

Wound Dressings ◽

Ftir Analysis ◽

Tissue Engineering Scaffolds ◽

Nanoscale Structures ◽

Low Frequency Ultrasound ◽

Chitosan Membranes ◽

The Stability

Naturally derived biopolymers have been widely used for biomedical applications such as drug carriers, wound dressings, and tissue engineering scaffolds. Chitosan is a typical polysaccharide of great interest due to its biocompatibility and film-formability. Chitosan membranes with controllable porous structures also have significant potential in membrane chromatography. Thus, the processing of membranes with porous nanoscale structures is of great importance, but it is also challenging and this has limited the application of these membranes to date. In this study, with the aid of a carefully selected surfactant, polyethyleneglycol stearate-40, chitosan membranes with a well controlled nanoscale structure were successfully prepared. Additional control over the membrane structure was obtained by exposing the suspension to high intensity, low frequency ultrasound. It was found that the concentration of chitosan/surfactant ratio and the ultrasound exposure conditions affect the structural features of the membranes. The stability of nanopores in the membrane was improved by intensive ultrasonication. Furthermore, the stability of the blended suspensions and the intermolecular interactions between chitosan and the surfactant were investigated using scanning electron microscope and Fourier transform infrared spectroscopy (FTIR) analysis, respectively. Hydrogen bonds and possible reaction sites for molecular interactions in the two polymers were also confirmed by FTIR analysis.

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Crystallization and preliminary X-ray crystallographic analysis of PBPD2 fromListeria monocytogenes

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x14005470 ◽

2014 ◽

Vol 70 (4) ◽

pp. 535-537 ◽

Cited By ~ 1

Author(s):

Hyung Jin Cha ◽

Jae-Hee Jeong ◽

Yeon-Gil Kim

Keyword(s):

Biosynthetic Pathway ◽

Crystallographic Analysis ◽

Diffusion Method ◽

Low Molecular Weight ◽

Molecular Replacement ◽

Orthorhombic Space Group ◽

Penicillin Binding Proteins ◽

Cell Parameters ◽

Replacement Method ◽

Molecular Replacement Method

Penicillin-binding proteins (PBPs), which mediate the peptidoglycan biosynthetic pathway in the bacterial cell wall, have been intensively investigated as a target for the design of antibiotics. In this study, PBPD2, a low-molecular-weight PBP encoded bylmo2812fromListeria monocytogenes, was overexpressed inEscherichia coli, purified and crystallized at 295 K using the sitting-drop vapour-diffusion method. The crystal belonged to the primitive orthorhombic space groupP212121, with unit-cell parametersa= 37.7,b= 74.7,c= 75.1 Å, and diffracted to 1.55 Å resolution. There was one molecule in the asymmetric unit. The preliminary structure was determined by the molecular-replacement method.

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Penicillin-binding proteins and carboxypeptidase/transpeptidase activities in Proteus vulgaris P18 and its penicillin-induced stable L-forms

Journal of Bacteriology ◽

10.1128/jb.152.3.1042-1048.1982 ◽

1982 ◽

Vol 152 (3) ◽

pp. 1042-1048

Author(s):

A Rousset ◽

M Nguyen-Distèche ◽

R Minck ◽

J M Ghuysen

Keyword(s):

Plasma Membrane ◽

Binding Proteins ◽

Binding Protein ◽

Penicillin Binding Protein ◽

Proteus Vulgaris ◽

Penicillin Binding Proteins

The originally penicillin-induced, wall-less stable L-forms of Proteus vulgaris P18, isolated by Tulasne in 1949 and since then cultured in he absence of penicillin, have kept the ability to synthesize the seven penicillin-binding proteins and the various DD- and LD-peptidase activities found in the parental bacteria and known to be involved in wall peptidoglycan metabolism. The stable L-forms, however, secrete during growth both the highly penicillin-sensitive, DD-carboxy-peptidase-transpeptidase penicillin-binding protein PBP4 (which in normal bacteria is relatively loosely bound to the plasma membrane) and the penicillin-insensitive LD-carboxypeptidase (which in normal bacteria is located in the periplasmic region).

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Structures of the ATP-fueled ClpXP proteolytic machine bound to protein substrate

eLife ◽

10.7554/elife.52774 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 24

Author(s):

Xue Fei ◽

Tristan A Bell ◽

Simon Jenni ◽

Benjamin M Stinson ◽

Tania A Baker ◽

...

Keyword(s):

Single Molecule ◽

Atp Hydrolysis ◽

Functional Results ◽

Structural Features ◽

Specific Protein ◽

Power Stroke ◽

E Coli ◽

Protein Substrates ◽

Biophysical Studies ◽

Sequential Models

ClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies of the E. coli enzyme that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel, as observed for other AAA+ proteases and protein-remodeling machines. Strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been inferred from these structural features for other AAA+ unfoldases, but biochemical and single-molecule biophysical studies indicate that ClpXP operates by a probabilistic mechanism in which five to eight residues are translocated for each ATP hydrolyzed. We propose structure-based models that could account for the functional results.

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Contribution of Hydrophobic Residues to the Stability of Ribonuclease A Chain Folding Initiation Site Mutants. A Comparison of Pressure and Temperature Induced Unfolding

Advances in High Pressure Bioscience and Biotechnology ◽

10.1007/978-3-642-60196-5_56 ◽

1999 ◽

pp. 251-254

Author(s):

J. Torrent ◽

J. P. Connelly ◽

M. G. Coll ◽

M. Ribό ◽

R. Lange ◽

...

Keyword(s):

Initiation Site ◽

Ribonuclease A ◽

Hydrophobic Residues ◽

Chain Folding ◽

A Chain ◽

The Stability

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Surface-Tension-Confined Channel with Biomimetic Microstructures for Unidirectional Liquid Spreading

Micromachines ◽

10.3390/mi11110978 ◽

2020 ◽

Vol 11 (11) ◽

pp. 978

Author(s):

Yi Zhang ◽

Yang Gan ◽

Liwen Zhang ◽

Deyuan Zhang ◽

Huawei Chen

Keyword(s):

Surface Tension ◽

Underlying Mechanism ◽

Structural Features ◽

Liquid Motion ◽

Water Separation ◽

Lab On Chip ◽

Liquid Spreading ◽

Oil Water Separation ◽

On Chip ◽

The Stability

Unidirectional liquid spreading without energy input is of significant interest for the broad applications in diverse fields such as water harvesting, drop transfer, oil–water separation and microfluidic devices. However, the controllability of liquid motion and the simplification of manufacturing process remain challenges. Inspired by the peristome of Nepenthes alata, a surface-tension-confined (STC) channel with biomimetic microcavities was fabricated facilely through UV exposure photolithography and partial plasma treatment. Perfect asymmetric liquid spreading was achieved by combination of microcavities and hydrophobic boundary, and the stability of pinning effect was demonstrated. The influences of structural features of microcavities on both liquid spreading and liquid pinning were investigated and the underlying mechanism was revealed. We also demonstrated the spontaneous unidirectional transport of liquid in 3D space and on tilting slope. In addition, through changing pits arrangement and wettability pattern, complex liquid motion paths and microreactors were realized. This work will open a new way for liquid manipulation and lab-on-chip applications.

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