scholarly journals Crystallization and structure analysis of the core motif of the Pks13 acyltransferase domain from Mycobacterium tuberculosis

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4728 ◽  
Author(s):  
Mingjing Yu ◽  
Chao Dou ◽  
Yijun Gu ◽  
Wei Cheng
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Target ◽  
Polyketide Synthase ◽  
Mycolic Acid ◽  
Type I ◽  
Core Motif ◽  
Conserved Residues ◽  
High Resolution Structure ◽  
Essential Enzyme ◽  
Resolution Structure

Type I polyketide synthase 13 (Pks13) is involved in the final step of the biosynthesis of mycolic acid in Mycobacterium tuberculosis. Recent articles have reported that Pks13 is an essential enzyme in the mycolic acid biosynthesis pathway, and it has been deeply studied as a drug target in Tuberculosis. We report a high-resolution structure of the acyltransferase (AT) domain of Pks13 at 2.59 Å resolution. Structural comparison with the full-length AT domain (PDB code, 3TZW, and 3TZZ) reveals a different orientation of the C-terminal helix and rearrangement of some conserved residues.

scholarly journals High-resolution structure determination of sub-100 kilodalton complexes using conventional cryo-EM

2018 ◽  
Author(s):  
Mark A. Herzik ◽  
Mengyu Wu ◽  
Gabriel C. Lander
Keyword(s):  
High Resolution ◽  
Structure Determination ◽  
Drug Target ◽  
Phase Plate ◽  
Biological Macromolecules ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
High Resolution Structure ◽  
Resolution Structure

Determining high-resolution structures of biological macromolecules with masses of less than 100 kilodaltons (kDa) has long been a goal of the cryo-electron microscopy (cryo-EM) community. While the Volta Phase Plate has enabled cryo-EM structure determination of biological specimens of this size range, use of this instrumentation is not yet fully automated and can present technical challenges. Here, we show that conventional defocus-based cryo-EM methodologies can be used to determine the high-resolution structures of specimens amassing less than 100 kDa using a transmission electron microscope operating at 200 keV coupled with a direct electron detector. Our ~2.9 Å structure of alcohol dehydrogenase (82 kDa) proves that bound ligands can be resolved with high fidelity, indicating that these methodologies can be used to investigate the molecular details of drug-target interactions. Our ~2.8 Å and ~3.2 Å resolution structures of methemoglobin demonstrate that distinct conformational states can be identified within a dataset for proteins as small as 64 kDa. Furthermore, we provide the first sub-nanometer cryo-EM structure of a protein smaller than 50 kDa.

scholarly journals 1.2 Å resolution crystal structure of the periplasmic aminotransferase PvdN fromPseudomonas aeruginosa

2016 ◽  
Vol 72 (5) ◽  
pp. 403-408 ◽  
Author(s):  
Eric J. Drake ◽  
Andrew M. Gulick
Keyword(s):  
Biosynthetic Pathway ◽  
Pyridoxal Phosphate ◽  
Positive Density ◽  
Type I ◽  
Structural Homology ◽  
Peptide Synthetase ◽  
High Resolution Structure ◽  
Multi Wavelength ◽  
Peptide Product ◽  
Resolution Structure

The Gram-negative pathogenPseudomonas aeruginosauses a nonribosomal peptide synthetase (NRPS) biosynthetic cluster for the production of a peptide siderophore. In addition to four multimodular NRPS proteins, the biosynthetic pathway also requires several additional enzymes involved in the production of nonproteinogenic amino acids and maturation of the peptide product. Among the proteins that are required for the final steps in pyoverdine synthesis is PvdN, a pyridoxal phosphate-dependent enzyme that catalyzes an uncharacterized step in pyoverdine production. This study reports the high-resolution structure of PvdN bound to a PLP cofactor solved by multi-wavelength anomalous dispersion (MAD). The PvdN model shows high structural homology to type I aspartate aminotransferases and also contains positive density that suggests an uncharacterized external aldimine.

The High-resolution Structure of LeuB (Rv2995c) from Mycobacterium tuberculosis

2005 ◽  
Vol 346 (1) ◽  
pp. 1-11 ◽  
Author(s):  
Rajesh Kumar Singh ◽  
Georgia Kefala ◽  
Robert Janowski ◽  
Christoph Mueller-Dieckmann ◽  
Jens-Peter von Kries ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Resolution ◽  
High Resolution Structure ◽  
Resolution Structure

scholarly journals INVESTIGATION OF ACCD3 GENE OF MYCOBACTERIUM TUBERCULOSIS IRAQI ISOLATES

2018 ◽  
Vol 11 (8) ◽  
pp. 208
Author(s):  
Asra'a A Abdul Jalil ◽  
Zahra M Al-khafaji ◽  
Mushtak T Al-ouqaili
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Target ◽  
Conventional Polymerase Chain Reaction ◽  
Mycolic Acid ◽  
Human Pathogens ◽  
Mycolic Acids ◽  
Pcr Product ◽  
Malonyl Coa ◽  
Polymerase Chain ◽  
Acetyl Coenzyme A Carboxylase

Objective: Mycobacterium tuberculosis, one of the deadliest human pathogens, causes several million new infections and about 2 million fatalities annually. The cell wall of M. tuberculosis is endowed with a highly impermeable, complex array of diverse lipids such as mycolic acids, which bestow the bacterium with not only virulence but also resistance to host immunity and antibiotics.Methods: Mycobacterial lipid metabolism has thus emerged as an attractive target for the design and development of novel antimycobacterial therapeutics. The first committed step in the biosynthesis of mycolic acid is the carboxylation of acetyl-CoA to malonyl-CoA which is catalyzed by acetyl-coenzyme A carboxylase carboxyl transferase beta subunit (accD3), a primer pairs were designed computationally and used for the amplification of accD3 gene using conventional polymerase chain reaction (PCR) and sequencing the PCR product and analyze the results.Results: Two sequences of the detection gene (LprM gene) and eight sequences of accD3 gene under study were deposited at NCBI – GenBank database with accession numbers (LC009881, LC009880.1, LC006979, LC008196, LC009412, LC009414, LC034168, LC038020, LC041163, and LC041368) and primer pairs deposited at Probe database/NCBI with accession number Pr032816836.Conclusion: AccD3 gene is a good drug target in MDR M. tuberculosis strains.

Regulation of the intersubunit ammonia tunnel in Mycobacterium tuberculosis glutamine-dependent NAD+ synthetase

2012 ◽  
Vol 443 (2) ◽  
pp. 417-426 ◽  
Author(s):  
Watchalee Chuenchor ◽  
Tzanko I. Doukov ◽  
Melissa Resto ◽  
Andrew Chang ◽  
Barbara Gerratana
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Active Site ◽  
Drug Target ◽  
Wild Type ◽  
Ammonia Transport ◽  
Site Variation ◽  
Ammonia Tunnel ◽  
Essential Enzyme ◽  
Nad Synthetase ◽  
Insight Into

Glutamine-dependent NAD+ synthetase is an essential enzyme and a validated drug target in Mycobacterium tuberculosis (mtuNadE). It catalyses the ATP-dependent formation of NAD+ from NaAD+ (nicotinic acid–adenine dinucleotide) at the synthetase active site and glutamine hydrolysis at the glutaminase active site. An ammonia tunnel 40 Å (1 Å=0.1 nm) long allows transfer of ammonia from one active site to the other. The enzyme displays stringent kinetic synergism; however, its regulatory mechanism is unclear. In the present paper, we report the structures of the inactive glutaminase C176A variant in an apo form and in three synthetase–ligand complexes with substrates (NaAD+/ATP), substrate analogue {NaAD+/AMP-CPP (adenosine 5′-[α,β-methylene]triphosphate)} and intermediate analogues (NaAD+/AMP/PPi), as well as the structure of wild-type mtuNadE in a product complex (NAD+/AMP/PPi/glutamate). This series of structures provides snapshots of the ammonia tunnel during the catalytic cycle supported also by kinetics and mutagenesis studies. Three major constriction sites are observed in the tunnel: (i) at the entrance near the glutaminase active site; (ii) in the middle of the tunnel; and (iii) at the end near the synthetase active site. Variation in the number and radius of the tunnel constrictions is apparent in the crystal structures and is related to ligand binding at the synthetase domain. These results provide new insight into the regulation of ammonia transport in the intermolecular tunnel of mtuNadE.

scholarly journals Modelling the active SARS-CoV-2 helicase complex as a basis for structure-based inhibitor design

2020 ◽  
Author(s):  
Dénes Berta ◽  
Magd Badaoui ◽  
Pedro J. Buigues ◽  
Sam Alexander Martino ◽  
Andrei V. Pisliakov ◽  
...  
Keyword(s):  
Drug Target ◽  
Md Simulation ◽  
Structural Information ◽  
Rna Helicase ◽  
Atomic Level ◽  
Virus Rna ◽  
High Resolution Structure ◽  
And Function ◽  
Structural Insights ◽  
Resolution Structure

ABSTRACTHaving claimed over 1 million lives worldwide to date, the ongoing COVID-19 pandemic has created one of the biggest challenges to develop an effective drug to treat infected patients. Among all the proteins expressed by the virus, RNA helicase is a fundamental protein for viral replication, and it is highly conserved among the coronaviridae family. To date, there is no high-resolution structure of helicase bound with ATP and RNA. We present here structural insights and molecular dynamics (MD) simulation results of the SARS-CoV-2 RNA helicase both in its apo form and in complex with its natural substrates. Our structural information of the catalytically competent helicase complex provides valuable insights for the mechanism and function of this enzyme at the atomic level, a key to develop specific inhibitors for this potential COVID-19 drug target.

scholarly journals Essentiality and functional analysis of type I and type III pantothenate kinases of Mycobacterium tuberculosis

Microbiology ◽  
2010 ◽  
Vol 156 (9) ◽  
pp. 2691-2701 ◽  
Author(s):  
Disha Awasthy ◽  
Anisha Ambady ◽  
Jyothi Bhat ◽  
Gulebahar Sheikh ◽  
Sudha Ravishankar ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Target ◽  
Structural Characteristics ◽  
Temperature Sensitive ◽  
Type I ◽  
Permissive Temperature ◽  
Extra Copy ◽  
Pantothenate Kinase ◽  
In The Wild

Pantothenate kinase, an essential enzyme in bacteria and eukaryotes, is involved in catalysing the first step of conversion of pantothenate to coenzyme A (CoA). Three isoforms (type I, II and III) of this enzyme have been reported from various organisms, which can be differentiated from each other on the basis of their biochemical and structural characteristics. Though most bacteria carry only one of the isoforms of pantothenate kinases, some of them possess two isoforms. The physiological relevance of the presence of two types of isozymes in a single organism is not clear. Mycobacterium tuberculosis, an intracellular pathogen, possesses two isoforms of pantothenate kinases (CoaA and CoaX) belonging to type I and III. In order to determine which pantothenate kinase is essential in mycobacteria, we performed gene inactivation of coaA and coaX of M. tuberculosis individually. It was found that coaA could only be inactivated in the presence of an extra copy of the gene, while coaX could be inactivated in the wild-type cells, proving that CoaA is the essential pantothenate kinase in M. tuberculosis. Additionally, the coaA gene of M. tuberculosis was able to complement a temperature-sensitive coaA mutant of Escherichia coli at a non-permissive temperature while coaX could not. The coaX deletion mutant showed no growth defects in vitro, in macrophages or in mice. Taken together, our data suggest that CoaX, which is essential in Bacillus anthracis and thus had been suggested to be a drug target in this organism, might not be a valid target in M. tuberculosis. We have established that the type I isoform, CoaA, is the essential pantothenate kinase in M. tuberculosis and thus can be explored as a drug target.

High-resolution structure determination by ALCHEMI

1983 ◽  
Vol 41 ◽  
pp. 178-181 ◽  
Author(s):  
Peter G. Self ◽  
Peter R. Buseck
Keyword(s):  
Site Occupancy ◽  
Real Space ◽  
Unit Cell ◽  
Bragg Angle ◽  
Electron Current ◽  
High Resolution Structure ◽  
Beam Incident ◽  
Crystallographic Planes ◽  
Electron Beam Incident ◽  
Resolution Structure

ALCHEMI (Atom Location by CHanneling Enhanced Microanalysis) enables the site occupancy of atoms in single crystals to be determined. In this article the fundamentals of the method for both EDS and EELS will be discussed. Unlike HRTEM, ALCHEMI does not place stringent resolution requirements on the microscope and, because EDS clearly distinguishes between elements of similar atomic number, it can offer some advantages over HRTEM. It does however, place certain constraints on the crystal. These constraints are: a) the sites of interest must lie on alternate crystallographic planes, b) the projected charge density on the alternate planes must be significantly different, and c) there must be at least one atomic species that lies solely on one of the planes.An electron beam incident on a crystal undergoes elastic scattering; in reciprocal space this is seen as a diffraction pattern and in real space this is a modulation of the electron current across the unit cell. When diffraction is strong (i.e., when the crystal is oriented near to the Bragg angle of a low-order reflection) the electron current at one point in the unit cell will differ significantly from that at another point.

Cathodoluminescence of Catalyst Crystallites

1982 ◽  
Vol 40 ◽  
pp. 664-665
Author(s):  
E.D. Boyes ◽  
P.L. Gai ◽  
D.B. Darby ◽  
C. Warwick
Keyword(s):  
Defect Density ◽  
Chemical Properties ◽  
Operating Conditions ◽  
Semiconductor Materials ◽  
Environmental Cell ◽  
High Resolution Structure ◽  
Commercially Important ◽  
Crystallographic Defects ◽  
Resolution Structure

The extended crystallographic defects introduced into some oxide catalysts under operating conditions may be a consequence and accommodation of the changes produced by the catalytic activity, rather than always being the origin of the reactivity. Operation without such defects has been established for the commercially important tellurium molybdate system. in addition it is clear that the point defect density and the electronic structure can both have a significant influence on the chemical properties and hence on the effectiveness (activity and selectivity) of the material as a catalyst. SEM/probe techniques more commonly applied to semiconductor materials, have been investigated to supplement the information obtained from in-situ environmental cell HVEM, ultra-high resolution structure imaging and more conventional AEM and EPMA chemical microanalysis.

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