scholarly journals The Structure and Function of Modular Escherichia coli O157:H7 Bacteriophage FTBEc1 endolysin, LysT84: Defining a New Endolysin Catalytic Subfamily

2021 ◽  
Author(s):  
Michael Love ◽  
David Coombes ◽  
Salim Ismail ◽  
Craig Billington ◽  
Renwick CJ Dobson

Bacteriophage endolysins degrade peptidoglycan and have been identified as antibacterial candidates to combat antimicrobial resistance. Considering the catalytic and structural diversity of endolysins, there is a paucity of structural data to inform how these enzymes work at the molecular level—key data that is needed to realize the potential of endolysin-based antibacterial agents. Here, we determine the atomic structure and define the enzymatic function of Escherichia coli O157:H7 phage FTEBc1 endolysin, LysT84. Bioinformatic analysis reveals that LysT84 is a modular endolysin, which is unusual for Gram-negative endolysins, comprising a peptidoglycan binding domain and an enzymatic domain. The crystal structure of LysT84 (2.99 Å) revealed a mostly α-helical protein with two domains connected by a linker region but packed together. LysT84 was determined to be a monomer in solution using analytical ultracentrifugation. Small-angle X-ray scattering data revealed that LysT84 is a flexible protein but does not have the expected bimodal P(r) function of a multidomain protein, suggesting that the domains of LysT84 pack closely creating a globular protein as seen in the crystal structure. Structural analysis reveals two key glutamate residues positioned on either side of the active site cavity; mutagenesis demonstrating these residues are critical for peptidoglycan degradation. Molecular dynamic simulations suggest that the enzymatically active domain is dynamic, allowing the appropriate positioning of these catalytic residues for hydrolysis of the β(1–4) bond. Overall, our study defines the structural basis for peptidoglycan degradation by LysT84 which supports rational engineering of related endolysins into effective antibacterial agents.

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1101
Author(s):  
Michael J. Love ◽  
David Coombes ◽  
Sarah H. Manners ◽  
Gayan S. Abeysekera ◽  
Craig Billington ◽  
...  

Bacteriophage-encoded endolysins have been identified as antibacterial candidates. However, the development of endolysins as mainstream antibacterial agents first requires a comprehensive biochemical understanding. This study defines the atomic structure and enzymatic function of Escherichia coli O157:H7 phage FAHEc1 endolysin, LysF1. Bioinformatic analysis suggests this endolysin belongs to the T4 Lysozyme (T4L)-like family of proteins and contains a highly conserved catalytic triad. We then solved the structure of LysF1 with x-ray crystallography to 1.71 Å. LysF1 was confirmed to exist as a monomer in solution by sedimentation velocity experiments. The protein architecture of LysF1 is conserved between T4L and related endolysins. Comparative analysis with related endolysins shows that the spatial orientation of the catalytic triad is conserved, suggesting the catalytic mechanism of peptidoglycan degradation is the same as that of T4L. Differences in the sequence illustrate the role coevolution may have in the evolution of this fold. We also demonstrate that by mutating a single residue within the hydrophobic core, the thermal stability of LysF1 can be increased by 9.4 °C without compromising enzymatic activity. Overall, the characterization of LysF1 provides further insight into the T4L-like class of endolysins. Our study will help advance the development of related endolysins as antibacterial agents, as rational engineering will rely on understanding mutable positions within this protein fold.


2017 ◽  
Vol 24 (2) ◽  
pp. 181-187 ◽  
Author(s):  
Yinliang Ma ◽  
Guohui Bai ◽  
Yaqi Cui ◽  
Jing Zhao ◽  
Zenglin Yuan ◽  
...  

2019 ◽  
Vol 167 (1) ◽  
pp. 1-14
Author(s):  
Koji Nagata ◽  
Akitoshi Okada ◽  
Jun Ohtsuka ◽  
Takatoshi Ohkuri ◽  
Yusuke Akama ◽  
...  

Abstract Loading the bacterial replicative helicase DnaB onto DNA requires a specific loader protein, DnaC/DnaI, which creates the loading-competent state by opening the DnaB hexameric ring. To understand the molecular mechanism by which DnaC/DnaI opens the DnaB ring, we solved 3.1-Å co-crystal structure of the interaction domains of Escherichia coli DnaB–DnaC. The structure reveals that one N-terminal domain (NTD) of DnaC interacts with both the linker helix of a DnaB molecule and the C-terminal domain (CTD) of the adjacent DnaB molecule by forming a three α-helix bundle, which fixes the relative orientation of the two adjacent DnaB CTDs. The importance of the intermolecular interface in the crystal structure was supported by the mutational data of DnaB and DnaC. Based on the crystal structure and other available information on DnaB–DnaC structures, we constructed a molecular model of the hexameric DnaB CTDs bound by six DnaC NTDs. This model suggested that the binding of a DnaC would cause a distortion in the hexameric ring of DnaB. This distortion of the DnaB ring might accumulate by the binding of up to six DnaC molecules, resulting in the DnaB ring to open.


2008 ◽  
Vol 13 (11) ◽  
pp. 3006-3016 ◽  
Author(s):  
Erumbi S. Rangarajan ◽  
Yunge Li ◽  
Pietro Iannuzzi ◽  
Ante Tocilj ◽  
Li-Wei Hung ◽  
...  

2016 ◽  
Vol 72 (2) ◽  
pp. 236-244 ◽  
Author(s):  
Zhen Chen ◽  
Li-Hong Zhan ◽  
Hai-Feng Hou ◽  
Zeng-Qiang Gao ◽  
Jian-Hua Xu ◽  
...  

InEscherichia coli, the Omp85 protein BamA and four lipoproteins (BamBCDE) constitute the BAM complex, which is essential for the assembly and insertion of outer membrane proteins into the outer membrane. Here, the crystal structure of BamB in complex with the POTRA3–4 domains of BamA is reported at 2.1 Å resolution. Based on this structure, the POTRA3 domain is associated with BamBviahydrogen-bonding and hydrophobic interactions. Structural and biochemical analysis revealed that the conserved residues Arg77, Glu127, Glu150, Ser167, Leu192, Leu194 and Arg195 of BamB play an essential role in interaction with the POTRA3 domain.


2006 ◽  
Vol 188 (15) ◽  
pp. 5606-5617 ◽  
Author(s):  
Ming-Ni Hung ◽  
Erumbi Rangarajan ◽  
Christine Munger ◽  
Guy Nadeau ◽  
Traian Sulea ◽  
...  

ABSTRACT Enterobacterial common antigen (ECA) is a polysaccharide found on the outer membrane of virtually all gram-negative enteric bacteria and consists of three sugars, N-acetyl-d-glucosamine, N-acetyl-d-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-d-galactose, organized into trisaccharide repeating units having the sequence →3)-α-d-Fuc4NAc-(1→4)-β-d-ManNAcA-(1→4)-α-d-GlcNAc-(1→. While the precise function of ECA is unknown, it has been linked to the resistance of Shiga-toxin-producing Escherichia coli (STEC) O157:H7 to organic acids and the resistance of Salmonella enterica to bile salts. The final step in the synthesis of 4-acetamido-4,6-dideoxy-d-galactose, the acetyl-coenzyme A (CoA)-dependent acetylation of the 4-amino group, is carried out by TDP-fucosamine acetyltransferase (WecD). We have determined the crystal structure of WecD in apo form at a 1.95-Å resolution and bound to acetyl-CoA at a 1.66-Å resolution. WecD is a dimeric enzyme, with each monomer adopting the GNAT N-acetyltransferase fold, common to a number of enzymes involved in acetylation of histones, aminoglycoside antibiotics, serotonin, and sugars. The crystal structure of WecD, however, represents the first structure of a GNAT family member that acts on nucleotide sugars. Based on this cocrystal structure, we have used flexible docking to generate a WecD-bound model of the acetyl-CoA-TDP-fucosamine tetrahedral intermediate, representing the structure during acetyl transfer. Our structural data show that WecD does not possess a residue that directly functions as a catalytic base, although Tyr208 is well positioned to function as a general acid by protonating the thiolate anion of coenzyme A.


2015 ◽  
Vol 71 (10) ◽  
pp. 1258-1263 ◽  
Author(s):  
J. Guo ◽  
P. Erskine ◽  
A. R. Coker ◽  
J. Gor ◽  
S. J. Perkins ◽  
...  

The enzyme 2,4′-dihydroxyacetophenone dioxygenase (DAD) catalyses the conversion of 2,4′-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid. This enzyme is a very unusual dioxygenase in that it cleaves a C—C bond in a substituent of the aromatic ring rather than within the ring itself. Whilst it has been shown that DAD is a tetramer in solution, the recently solved crystal structure of theAlcaligenessp. 4HAP enzyme was in fact dimeric rather than tetrameric. Since the use of limited chymotrypsinolysis, which apparently results in removal of the first 20 or so N-terminal residues of DAD, was necessary for crystallization of the protein, it was investigated whether this was responsible for the change in its oligomerization state. Gel-filtration and analytical ultracentrifugation studies were conducted, which confirmed that chymotrypsinolysed DAD has an apparent molecular weight of around 40 kDa, corresponding to a dimer. In contrast, the native enzyme has a molecular weight in the 70–80 kDa region, as expected for the tetramer. The structural basis for tetramerization has been investigated by the use of several docking servers, and the results are remarkably consistent with the tetrameric structure of a homologous cupin protein fromRalstonia eutropha(PDB entry 3ebr).


2005 ◽  
Vol 61 (2) ◽  
pp. 454-459 ◽  
Author(s):  
Stephane Raymond ◽  
Ante Tocilj ◽  
Eunice Ajamian ◽  
Yunge Li ◽  
Ming-Ni Hung ◽  
...  

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