Structural and Biochemical Studies of Dihydrofolate Reductase from Streptococcus pyogenes as a Target for Antifolate Antibiotics

ABSTRACTStreptococcus pyogenes, a beta-hemolytic bacterium, causes a wide spectrum of infections in human including pharyngitis, tonsillitis, scarlet fever, rheumatic fever, and necrotizing fasciitis. Streptococcal infections can also exist as co-infection with methicillin resistant Staphylococcus aureus (MRSA). Trimethoprim-sulfamethoxazole (TMP-SMX) combination has been used for treatment of S. pyogenes and MRSA co-infection. However, resistance to TMP, an inhibitor of dihydrofolate reductase enzyme (DHFR), has challenged the efficacy of TMP-SMX combination. We explored the activity of a series of novel DHFR inhibitors against S. pyogenes. This study identified potent inhibitors of DHFR enzyme from S. pyogenes with excellent inhibitory activity against the growth of the live bacteria. We determined, for the first time, the crystal structure of S. pyogenes DHFR which provides structural insights into design and development of antifolate agents against this global pathogen.

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The full-length Streptococcus pneumoniae major pilin RrgB crystallizes in a fibre-like structure, which presents the D1 isopeptide bond and provides details on the mechanism of pilus polymerization

Biochemical Journal ◽

10.1042/bj20111397 ◽

2012 ◽

Vol 441 (3) ◽

pp. 833-843 ◽

Cited By ~ 25

Author(s):

Lamya El Mortaji ◽

Carlos Contreras-Martel ◽

Monica Moschioni ◽

Ilaria Ferlenghi ◽

Clothilde Manzano ◽

...

Keyword(s):

Crystal Structure ◽

Streptococcus Pneumoniae ◽

Crystal Packing ◽

Full Length ◽

Intermolecular Bond ◽

Isopeptide Bond ◽

Live Bacteria ◽

Sorting Motif ◽

Density Map ◽

First Time

RrgB is the major pilin which forms the pneumococcal pilus backbone. We report the high-resolution crystal structure of the full-length form of RrgB containing the IPQTG sorting motif. The RrgB fold is organized into four distinct domains, D1–D4, each of which is stabilized by an isopeptide bond. Crystal packing revealed a head-to-tail organization involving the interaction of the IPQTG motif into the D1 domain of two successive RrgB monomers. This fibrillar assembly, which fits into the electron microscopy density map of the native pilus, probably induces the formation of the D1 isopeptide bond as observed for the first time in the present study, since neither in published structures nor in soluble RrgB produced in Escherichia coli or in Streptococcus pneumoniae is the D1 bond present. Experiments performed in live bacteria confirmed that the intermolecular bond linking the RrgB subunits takes place between the IPQTG motif of one RrgB subunit and the Lys183 pilin motif residue of an adjacent RrgB subunit. In addition, we present data indicating that the D1 isopeptide bond is involved in RrgB stabilization. In conclusion, the crystal RrgB fibre is a compelling model for deciphering the molecular details required to generate the pneumococcal pilus.

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The clinical value of ficolin-3 gene polymorphism in rheumatic heart disease. An Egyptian adolescents study

BMC Research Notes ◽

10.1186/s13104-021-05450-w ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Maher H. Gomaa ◽

Emad Gamil Khidr ◽

Ahmed Elshafei ◽

Hala S. Hamza ◽

Aya M. Fattouh ◽

...

Keyword(s):

Innate Immunity ◽

Heart Disease ◽

Gene Polymorphism ◽

Rheumatic Heart Disease ◽

Streptococcus Pyogenes ◽

Gene Polymorphisms ◽

Clinical Value ◽

Homozygous Genotype ◽

First Time ◽

Rheumatic Heart

Abstract Objective Ficolin-3 is one of the innate immunity molecules that was thought to play a pivotal role in Streptococcus pyogenes autoimmunity and its complications; rheumatic fever (RF) and rheumatic heart disease (RHD). We aimed to disclose if there is an association between ficolin-3 (FCN3) gene polymorphisms (rs4494157 and rs10794501) and RF with or without RHD for the first time in Egyptian adolescents. Results Serum ficolin-3 level was significantly elevated in patients suffering from RF with and without RHD in comparison with control. Regarding FCN3 gene (rs4494157) polymorphism, a significant correlation was found between the A allele and the susceptibility to RF with or without RHD (OR = 2.93, P = 0.0002 and OR = 2.23, P = 0.008 respectively). Besides, AA homozygous genotype showed a significant association with RHD risk (OR = 3.47, P = 0.026). Patients carrying the A allele (CA + AA) had significantly higher serum ficolin-3 than those carrying the CC genotype (P ˂ 0.0001). While the frequency of (rs10794501) polymorphism revealed no significant differences between the controls and RF patients with or without RHD (OR = 1.43, P = 0.261 and OR = 1.48, P = 0.208 respectively).

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Investigation of Solubility Behavior of Canagliflozin Hydrate Crystals Combining Crystallographic and Hirshfeld Surface Calculations

Molecules ◽

10.3390/molecules26020298 ◽

2021 ◽

Vol 26 (2) ◽

pp. 298

Author(s):

Yefen Zhu ◽

Yanlei Kang ◽

Ling Zhu ◽

Kaxi Yu ◽

Shuai Chen ◽

...

Keyword(s):

Crystal Structure ◽

Intermolecular Forces ◽

Hirshfeld Surface ◽

Reversible Inhibitor ◽

Solubility Behavior ◽

Hydrogen Bond Networks ◽

Solvent Molecules ◽

First Time ◽

Better Than

Canagliflozin (CG) was a highly effective, selective and reversible inhibitor of sodium-dependent glucose co-transporter 2 developed for the treatment of type 2 diabetes mellitus. The crystal structure of CG monohydrate (CG-H2O) was reported for the first time while CG hemihydrate (CG-Hemi) had been reported in our previous research. Solubility and dissolution rate results showed that the solubility of CG-Hemi was 1.4 times higher than that of CG-H2O in water and hydrochloric acid solution, and the dissolution rates of CG-Hemi were more than 3 folds than CG-H2O in both solutions. Hirshfeld surface analysis showed that CG-H2O had stronger intermolecular forces than CG-Hemi, and water molecules in CG-H2O participated three hydrogen bonds, forming hydrogen bond networks. These crystal structure features might make it more difficult for solvent molecules to dissolve CG-H2O than CG-Hemi. All these analyses might explain why the dissolution performance of CG-Hemi was better than CG-H2O. This work provided an approach to predict the dissolution performance of the drug based on its crystal structure.

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Synthesis, crystal structure and photoluminescence properties of Eu2+-activated RbCaGd(PO4)2 phosphors

RSC Advances ◽

10.1039/c5ra25124a ◽

2016 ◽

Vol 6 (14) ◽

pp. 11211-11217 ◽

Cited By ~ 4

Author(s):

Xue Chen ◽

Qidi Wang ◽

Fengzhu Lv ◽

Paul K. Chu ◽

Yihe Zhang

Keyword(s):

Crystal Structure ◽

Sol Gel ◽

Dipole Interaction ◽

Concentration Quenching ◽

Photoluminescence Properties ◽

Gel Method ◽

Sol Gel Method ◽

First Time

RbCaGd(PO4)2:Eu2+ was prepared by the Pechini-type sol–gel method. The crystal structure was determined in the first time. The dipole–dipole interaction plays a major role in the mechanism of concentration quenching of Eu2+ in this phosphor.

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Structural insights into the novel ARM-repeat protein CTNNBL1 and its association with the hPrp19–CDC5L complex

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s139900471303318x ◽

2014 ◽

Vol 70 (3) ◽

pp. 780-788 ◽

Cited By ~ 2

Author(s):

Jae-Woo Ahn ◽

Sangwoo Kim ◽

Eun-Jung Kim ◽

Yeo-Jin Kim ◽

Kyung-Jin Kim

Keyword(s):

Crystal Structure ◽

The Novel ◽

Molecular Architecture ◽

Repeat Protein ◽

Binding Partner ◽

Catalytic Activation ◽

Repeat Structure ◽

Repeat Domain ◽

Importin Α ◽

Structural Insights

The hPrp19–CDC5L complex plays a crucial role during human pre-mRNA splicing by catalytic activation of the spliceosome. In order to elucidate the molecular architecture of the hPrp19–CDC5L complex, the crystal structure of CTNNBL1, one of the major components of this complex, was determined. Unlike canonical ARM-repeat proteins such as β-catenin and importin-α, CTNNBL1 was found to contain a twisted and extended ARM-repeat structure at the C-terminal domain and, more importantly, the protein formed a stable dimer. A highly negatively charged patch formed in the N-terminal ARM-repeat domain of CTNNBL1 provides a binding site for CDC5L, a binding partner of the protein in the hPrp19–CDC5L complex, and these two proteins form a complex with a stoichiometry of 2:2. These findings not only present the crystal structure of a novel ARM-repeat protein, CTNNBL1, but also provide insights into the detailed molecular architecture of the hPrp19–CDC5L complex.

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Specificity of Human Sulfiredoxin for Reductant and Peroxiredoxin Oligomeric State

Antioxidants ◽

10.3390/antiox10060946 ◽

2021 ◽

Vol 10 (6) ◽

pp. 946

Author(s):

Tom E. Forshaw ◽

Julie A. Reisz ◽

Kimberly J. Nelson ◽

Rajesh Gumpena ◽

J. Reed Lawson ◽

...

Keyword(s):

Crystal Structure ◽

Antioxidant Enzymes ◽

Hydrogen Sulfide ◽

Active Site ◽

Relative Efficacy ◽

Oligomeric Structure ◽

Cellular Functions ◽

Oligomeric State ◽

Structural Requirements ◽

First Time

Human peroxiredoxins (Prx) are a family of antioxidant enzymes involved in a myriad of cellular functions and diseases. During the reaction with peroxides (e.g., H2O2), the typical 2-Cys Prxs change oligomeric structure between higher order (do)decamers and disulfide-linked dimers, with the hyperoxidized inactive state (-SO2H) favoring the multimeric structure of the reduced enzyme. Here, we present a study on the structural requirements for the repair of hyperoxidized 2-Cys Prxs by human sulfiredoxin (Srx) and the relative efficacy of physiological reductants hydrogen sulfide (H2S) and glutathione (GSH) in this reaction. The crystal structure of the toroidal Prx1-Srx complex shows an extended active site interface. The loss of this interface within engineered Prx2 and Prx3 dimers yielded variants more resistant to hyperoxidation and repair by Srx. Finally, we reveal for the first time Prx isoform-dependent use of and potential cooperation between GSH and H2S in supporting Srx activity.

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Structural insights into the substrate specificity of a glycoside hydrolase family 5 lichenase from Caldicellulosiruptor sp. F32

Biochemical Journal ◽

10.1042/bcj20170328 ◽

2017 ◽

Vol 474 (20) ◽

pp. 3373-3389 ◽

Cited By ~ 9

Author(s):

Dong-Dong Meng ◽

Xi Liu ◽

Sheng Dong ◽

Ye-Fei Wang ◽

Xiao-Qing Ma ◽

...

Keyword(s):

Crystal Structure ◽

Substrate Specificity ◽

Glycoside Hydrolase ◽

Complex Structure ◽

Dynamics Simulation ◽

Structural Basis ◽

Glycoside Hydrolase Family ◽

Substrate Selectivity ◽

Glucose Residue ◽

Structural Insights

Glycoside hydrolase (GH) family 5 is one of the largest GH families with various GH activities including lichenase, but the structural basis of the GH5 lichenase activity is still unknown. A novel thermostable lichenase F32EG5 belonging to GH5 was identified from an extremely thermophilic bacterium Caldicellulosiruptor sp. F32. F32EG5 is a bi-functional cellulose and a lichenan-degrading enzyme, and exhibited a high activity on β-1,3-1,4-glucan but side activity on cellulose. Thin-layer chromatography and NMR analyses indicated that F32EG5 cleaved the β-1,4 linkage or the β-1,3 linkage while a 4-O-substitued glucose residue linked to a glucose residue through a β-1,3 linkage, which is completely different from extensively studied GH16 lichenase that catalyses strict endo-hydrolysis of the β-1,4-glycosidic linkage adjacent to a 3-O-substitued glucose residue in the mixed-linked β-glucans. The crystal structure of F32EG5 was determined to 2.8 Å resolution, and the crystal structure of the complex of F32EG5 E193Q mutant and cellotetraose was determined to 1.7 Å resolution, which revealed that the exit subsites of substrate-binding sites contribute to both thermostability and substrate specificity of F32EG5. The sugar chain showed a sharp bend in the complex structure, suggesting that a substrate cleft fitting to the bent sugar chains in lichenan is a common feature of GH5 lichenases. The mechanism of thermostability and substrate selectivity of F32EG5 was further demonstrated by molecular dynamics simulation and site-directed mutagenesis. These results provide biochemical and structural insights into thermostability and substrate selectivity of GH5 lichenases, which have potential in industrial processes.

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New Principles of the Substructure Development in Metal Materials under Plastic Deformation, Revealed by Advanced X-Ray Methods

Materials Science Forum ◽

10.4028/www.scientific.net/msf.443-444.259 ◽

2004 ◽

Vol 443-444 ◽

pp. 259-262 ◽

Cited By ~ 1

Author(s):

Yuriy Perlovich ◽

Margarita Isaenkova

Keyword(s):

Wide Spectrum ◽

Residual Deformation ◽

Ray Method ◽

Computer Data ◽

Rolled Material ◽

X Ray ◽

Pole Figures ◽

Metal Materials ◽

First Time ◽

Ray Methods

The substructure inhomogeneity of real textured metal materials was studied by use of the X-ray method of Generalized Pole Figures and the computer data treatment. Main regularities of substructure inhomogeneity were revealed for the first time. Substructure conditions of grains in rolled material form an extremely wide spectrum and vary by passing from texture maxima to texture minima, where residual deformation effects are most significant. The distribution of residual elastic microstrains in the orientational space of rolled material shows the distinct system.

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EFFECT OF EXTRACHROMOSOMAL ELEMENTS OF HEREDITY ON TOXIC PROPERTIES OF YERSINIA PESTIS

Journal of microbiology epidemiology immunobiology ◽

10.36233/0372-9311-2017-2-28-33 ◽

2017 ◽

pp. 28-33 ◽

Cited By ~ 1

Author(s):

G. V. Demidova ◽

E. P. Sokolova ◽

V. P. Zyuzina ◽

V. A. Rykova ◽

I. V. Morozova ◽

...

Keyword(s):

Yersinia Pestis ◽

Active Form ◽

Infectious Agent ◽

Toxic Shock ◽

Toxic Activity ◽

Virulent Strains ◽

Extrachromosomal Elements ◽

Live Bacteria ◽

First Time

Aim. Elucidation of the role of extrachromosomal elements of heredity in manifestations of toxic properties of Yersinia pestis. Materials and methods. The study was carried out in vaccine strain Y. pestis EV76 (pMTl, pCDl, pPCPl) and non-plasmid variants of vaccine EV76 (pMTl\ pCDl', pPCPl') and virulent 231 (рМТГ, pCDl’, pPCPl') strains of Y. pestis. Presence of functionally active form of lipopolysaccharide (LPS) in the incubation medium of the bacteria was evaluated via toxicity of supernatant of Y. pestis for intact animals (infection-toxic shock) and mice sensitized by D-GalN. Results. 37°C cultures of Y. pestis EV76 containing a full amount of plasmids were established to release LPS into the environment. Non-plasmid variants of both vaccine and virulent strains of Y. pestis pMTl', pCD Г, рРСР 1 do not have this ability. Separation of LPS from cell wall was detected in live bacteria of plague infectious agent. This process is assumed to be coupled with translocation of proteins coded by pMTl, pCDl, pPCPl plasmids from the cell into the environment. Conclusion. Functional interconnection between extrachromosomal elements of heredity and toxic activity of Y. pestis LPS is established for the first time.

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Gatorbulin-1, a distinct cyclodepsipeptide chemotype, targets a seventh tubulin pharmacological site

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2021847118 ◽

2021 ◽

Vol 118 (9) ◽

pp. e2021847118

Author(s):

Susan Matthew ◽

Qi-Yin Chen ◽

Ranjala Ratnayake ◽

Charles S. Fermaintt ◽

Daniel Lucena-Agell ◽

...

Keyword(s):

Total Synthesis ◽

Wide Spectrum ◽

Targeted Chemotherapy ◽

Structural Determination ◽

Marine Cyanobacterium ◽

Microtubule Stabilization ◽

Biochemical Studies ◽

Β Tubulin

Tubulin-targeted chemotherapy has proven to be a successful and wide spectrum strategy against solid and liquid malignancies. Therefore, new ways to modulate this essential protein could lead to new antitumoral pharmacological approaches. Currently known tubulin agents bind to six distinct sites at α/β-tubulin either promoting microtubule stabilization or depolymerization. We have discovered a seventh binding site at the tubulin intradimer interface where a novel microtubule-destabilizing cyclodepsipeptide, termed gatorbulin-1 (GB1), binds. GB1 has a unique chemotype produced by a marine cyanobacterium. We have elucidated this dual, chemical and mechanistic, novelty through multidimensional characterization, starting with bioactivity-guided natural product isolation and multinuclei NMR-based structure determination, revealing the modified pentapeptide with a functionally critical hydroxamate group; and validation by total synthesis. We have investigated the pharmacology using isogenic cancer cell screening, cellular profiling, and complementary phenotypic assays, and unveiled the underlying molecular mechanism by in vitro biochemical studies and high-resolution structural determination of the α/β-tubulin−GB1 complex.

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