scholarly journals The structure of Mycobacterium tuberculosis heme-degrading protein, MhuD, in complex with product

2019 ◽  
Author(s):  
Alex Chao ◽  
Kalistyn H. Burley ◽  
Paul J. Sieminski ◽  
David L. Mobley ◽  
Celia W. Goulding
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Complex Structure ◽  
Protein Product ◽  
Wild Type ◽  
Heme Oxygenases ◽  
High Affinity ◽  
Type Protein ◽  
Heme Structure ◽  
Α Helix ◽  
Dynamics Simulations

AbstractMycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, requires iron for survival. In Mtb, MhuD is the cytosolic protein that degrades imported heme. MhuD is distinct, both in sequence and structure, from canonical heme oxygenases (HOs) but homologous with IsdG-type proteins. Canonical HO is found mainly in eukaryotes, while IsdG-type proteins are predominantly found in prokaryotes including pathogens. While there are several published structures of MhuD and other IsdG-type proteins in complex with heme substrate, no structures have been reported of IsdG-type proteins in complex with product, unlike HOs. We recently showed that the Mtb variant MhuD-R26S produces biliverdin IXα (αBV) rather than the wild-type (WT) mycobilin isomers as product. Given that mycobilin and other IsdG-type protein products like staphylobilin are difficult to isolate in quantities sufficient for structure determination, here we use the MhuD-R26S variant and its product αBV as a proxy to study the IsdG-type protein/product complex. First we show that αBV has nanomolar affinity for MhuD and the R26S variant. Second we determined the MhuD-R26S-αBV complex structure to 2.5 Å, which reveals two notable features (1) two αBV molecules bound per active site and (2) a new α-helix (α3) as compared with the MhuD-heme structure. Finally, by molecular dynamics simulations we show that α3 is stable with the proximal αBV alone. MhuD’s high affinity for its product and structural and electrostatic changes that accompany substrate turnover suggest that there is an unidentified protein that is responsible for product extraction from MhuD and other IsdG-type proteins.

Conformational Changes of the Wild-Type hIAPP and the S20P Mutant in Water Revealed by Molecular Dynamics Simulations

2011 ◽  
Vol 396-398 ◽  
pp. 1554-1557
Author(s):  
Mian Wang ◽  
Jian Yi Wang
Keyword(s):  
Molecular Dynamics ◽  
Hydrogen Bond ◽  
Conformational Changes ◽  
Amyloid Fibrils ◽  
Helical Structure ◽  
Wild Type ◽  
Hydrogen Bond Interaction ◽  
Hydrogen Bond Networks ◽  
Α Helix ◽  
Dynamics Simulations

Conformational changes of wild-type (WT) hIAPP and the S20P mutant in explicit water are investigated using molecular dynamics. In the whole simulation, WT shows compacter structure and has more hydrogen-bond networks than S20P. The residues 14-18 in WT is always maintained as a helical structure which is stabilized by the hydrogen bond between Ser20 and NH group of His18, and the other regions in WT partially loosen from α-helix structures into the coil structures. The S20P mutant in a shortage of hydrogen-bond interaction unfolds faster than WT. This work provides insight into the specific conformation of IAPP which is associated with the generation of amyloid fibrils.

scholarly journals Improving the Thermostability and Activity of Transaminase From Aspergillus terreus by Charge-Charge Interaction

2021 ◽  
Vol 9 ◽  
Author(s):  
Jia-Ren Cao ◽  
Fang-Fang Fan ◽  
Chang-Jiang Lv ◽  
Hong-Peng Wang ◽  
Ye Li ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Aspergillus Terreus ◽  
Three Dimensional ◽  
Fold Increase ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Wild Type ◽  
Enzyme Thermal Stability ◽  
Α Helix ◽  
Dynamics Simulations

Transaminases that promote the amination of ketones into amines are an emerging class of biocatalysts for preparing a series of drugs and their intermediates. One of the main limitations of (R)-selective amine transaminase from Aspergillus terreus (At-ATA) is its weak thermostability, with a half-life (t1/2) of only 6.9 min at 40°C. To improve its thermostability, four important residue sites (E133, D224, E253, and E262) located on the surface of At-ATA were identified using the enzyme thermal stability system (ETSS). Subsequently, 13 mutants (E133A, E133H, E133K, E133R, E133Q, D224A, D224H, D224K, D224R, E253A, E253H, E253K, and E262A) were constructed by site-directed mutagenesis according to the principle of turning the residues into opposite charged ones. Among them, three substitutions, E133Q, D224K, and E253A, displayed higher thermal stability than the wild-type enzyme. Molecular dynamics simulations indicated that these three mutations limited the random vibration amplitude in the two α-helix regions of 130–135 and 148–158, thereby increasing the rigidity of the protein. Compared to the wild-type, the best mutant, D224K, showed improved thermostability with a 4.23-fold increase in t1/2 at 40°C, and 6.08°C increase in T5010. Exploring the three-dimensional structure of D224K at the atomic level, three strong hydrogen bonds were added to form a special “claw structure” of the α-helix 8, and the residues located at 151–156 also stabilized the α-helix 9 by interacting with each other alternately.

scholarly journals Mutations in fbiD (Rv2983) as a Novel Determinant of Resistance to Pretomanid and Delamanid in Mycobacterium tuberculosis

2020 ◽  
Vol 65 (1) ◽  
pp. e01948-20
Author(s):  
Dalin Rifat ◽  
Si-Yang Li ◽  
Thomas Ioerger ◽  
Keshav Shah ◽  
Jean-Philippe Lanoix ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clinical Evidence ◽  
Clinical Samples ◽  
Loss Of Function ◽  
Wild Type ◽  
Content Type ◽  
Solid Media ◽  
High Level ◽  
Level Resistance

ABSTRACTThe nitroimidazole prodrugs delamanid and pretomanid comprise one of only two new antimicrobial classes approved to treat tuberculosis (TB) in 50 years. Prior in vitro studies suggest a relatively low barrier to nitroimidazole resistance in Mycobacterium tuberculosis, but clinical evidence is limited to date. We selected pretomanid-resistant M. tuberculosis mutants in two mouse models of TB using a range of pretomanid doses. The frequency of spontaneous resistance was approximately 10−5 CFU. Whole-genome sequencing of 161 resistant isolates from 47 mice revealed 99 unique mutations, of which 91% occurred in 1 of 5 genes previously associated with nitroimidazole activation and resistance, namely, fbiC (56%), fbiA (15%), ddn (12%), fgd (4%), and fbiB (4%). Nearly all mutations were unique to a single mouse and not previously identified. The remaining 9% of resistant mutants harbored mutations in Rv2983 (fbiD), a gene not previously associated with nitroimidazole resistance but recently shown to be a guanylyltransferase necessary for cofactor F420 synthesis. Most mutants exhibited high-level resistance to pretomanid and delamanid, although Rv2983 and fbiB mutants exhibited high-level pretomanid resistance but relatively small changes in delamanid susceptibility. Complementing an Rv2983 mutant with wild-type Rv2983 restored susceptibility to pretomanid and delamanid. By quantifying intracellular F420 and its precursor Fo in overexpressing and loss-of-function mutants, we provide further evidence that Rv2983 is necessary for F420 biosynthesis. Finally, Rv2983 mutants and other F420H2-deficient mutants displayed hypersusceptibility to some antibiotics and to concentrations of malachite green found in solid media used to isolate and propagate mycobacteria from clinical samples.

scholarly journals Allelic Exchange and Mutant Selection Demonstrate that Common Clinical embCAB Gene Mutations Only Modestly Increase Resistance to Ethambutol in Mycobacterium tuberculosis

2009 ◽  
Vol 54 (1) ◽  
pp. 103-108 ◽  
Author(s):  
Hassan Safi ◽  
Robert D. Fleischmann ◽  
Scott N. Peterson ◽  
Marcus B. Jones ◽  
Behnam Jarrahi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
In Vitro Selection ◽  
Gene Mutations ◽  
Wild Type ◽  
Mutant Selection ◽  
Preferential Growth ◽  
Allelic Exchange ◽  
High Level ◽  
Isogenic Mutants

ABSTRACT Mutations within codon 306 of the Mycobacterium tuberculosis embB gene modestly increase ethambutol (EMB) MICs. To identify other causes of EMB resistance and to identify causes of high-level resistance, we generated EMB-resistant M. tuberculosis isolates in vitro and performed allelic exchange studies of embB codon 406 (embB406) and embB497 mutations. In vitro selection produced mutations already identified clinically in embB306, embB397, embB497, embB1024, and embC13, which result in EMB MICs of 8 or 14 μg/ml, 5 μg/ml, 12 μg/ml, 3 μg/ml, and 4 μg/ml, respectively, and mutations at embB320, embB324, and embB445, which have not been identified in clinical M. tuberculosis isolates and which result in EMB MICs of 8 μg/ml, 8 μg/ml, and 2 to 8 μg/ml, respectively. To definitively identify the effect of the common clinical embB497 and embB406 mutations on EMB susceptibility, we created a series of isogenic mutants, exchanging the wild-type embB497 CAG codon in EMB-susceptible M. tuberculosis strain 210 for the embB497 CGG codon and the wild-type embB406 GGC codon for either the embB406 GCC, embB406 TGC, embB406 TCC, or embB406 GAC codon. These new mutants showed 6-fold and 3- to 3.5-fold increases in the EMB MICs, respectively. In contrast to the embB306 mutants, the isogenic embB497 and embB406 mutants did not have preferential growth in the presence of isoniazid or rifampin (rifampicin) at their MICs. These results demonstrate that individual embCAB mutations confer low to moderate increases in EMB MICs. Discrepancies between the EMB MICs of laboratory mutants and clinical M. tuberculosis strains with identical mutations suggest that clinical EMB resistance is multigenic and that high-level EMB resistance requires mutations in currently unknown loci.

scholarly journals Exploration of the cofactor specificity of wild-type phosphite dehydrogenase and its mutant using molecular dynamics simulations

RSC Advances ◽  
2021 ◽  
Vol 11 (24) ◽  
pp. 14527-14533
Author(s):  
Kunlu Liu ◽  
Min Wang ◽  
Yubo Zhou ◽  
Hongxiang Wang ◽  
Yudong Liu ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulations ◽  
Wild Type ◽  
Cofactor Specificity ◽  
Phosphite Dehydrogenase ◽  
Dynamics Simulations

Phosphite dehydrogenase (Pdh) catalyzes the NAD-dependent oxidation of phosphite to phosphate with the formation of NADH.

scholarly journals Mutations of folC cause increased susceptibility to sulfamethoxazole in Mycobacterium tuberculosis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ruiqi Wang ◽  
Kun Li ◽  
Jifang Yu ◽  
Jiaoyu Deng ◽  
Yaokai Chen
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Clinical Isolates ◽  
Aminobenzoic Acid ◽  
Wild Type ◽  
Dihydropteroate Synthase ◽  
Expression Levels ◽  
Encoding Gene ◽  
Increased Susceptibility

AbstractPrevious studies showed that mutation of folC caused decreased expression of the dihydropteroate synthase encoding gene folP2 in Mycobacterium tuberculosis (M. tuberculosis). We speculated that mutation of folC in M. tuberculosis might affect the susceptibility to sulfamethoxazole (SMX). To prove this, 53 clinical isolates with folC mutations were selected and two folC mutants (I43A, I43T) were constructed based on M. tuberculosis H37Ra. The results showed that 42 of the 53 clinical isolates (79.2%) and the two lab-constructed folC mutants were more sensitive to SMX. To probe the mechanism by which folC mutations make M. tuberculosis more sensitive to SMX, folP2 was deleted in H37Ra, and expression levels of folP2 were compared between H37Ra and the two folC mutants. Although deletion of folP2 resulted in increased susceptibility to SMX, no difference in folP2 expression was observed. Furthermore, production levels of para-aminobenzoic acid (pABA) were compared between the folC mutants and the wild-type strain, and results showed that folC mutation resulted in decreased production of pABA. Taken together, we show that folC mutation leads to decreased production of pABA in M. tuberculosis and thus affects its susceptibility to SMX, which broadens our understanding of mechanisms of susceptibilities to antifolates in this bacterium.

scholarly journals Evaluation of wild-type MIC distributions as a tool for determination of clinical breakpoints for Mycobacterium tuberculosis

2009 ◽  
Vol 64 (4) ◽  
pp. 786-793 ◽  
Author(s):  
T. Schon ◽  
P. Jureen ◽  
C. G. Giske ◽  
E. Chryssanthou ◽  
E. Sturegard ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Wild Type ◽  
Clinical Breakpoints

scholarly journals Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs

2021 ◽  
Author(s):  
Amit Ketkar ◽  
Lane Smith ◽  
Callie Johnson ◽  
Alyssa Richey ◽  
Makayla Berry ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mutation Frequency ◽  
Control Sequence ◽  
Wild Type ◽  
Binding Properties ◽  
High Affinity ◽  
G4 Dna ◽  
G Quadruplex ◽  
Forward Mutagenesis

Abstract We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.

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