scholarly journals Structural Insights into a Bifunctional Peptide Methionine Sulfoxide Reductase MsrA/B Fusion Protein from Helicobacter pylori

Antioxidants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 389
Author(s):  
Sulhee Kim ◽  
Kitaik Lee ◽  
Sun-Ha Park ◽  
Geun-Hee Kwak ◽  
Min Seok Kim ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Helicobacter Pylori ◽  
Fusion Protein ◽  
Pathogenic Bacteria ◽  
Catalytic Efficiency ◽  
Salt Bridge ◽  
Methionine Sulfoxide ◽  
Methionine Sulfoxide Reductase ◽  
Salt Bridges ◽  
Linker Region

Methionine sulfoxide reductase (Msr) is a family of enzymes that reduces oxidized methionine and plays an important role in the survival of bacteria under oxidative stress conditions. MsrA and MsrB exist in a fusion protein form (MsrAB) in some pathogenic bacteria, such as Helicobacter pylori (Hp), Streptococcus pneumoniae, and Treponema denticola. To understand the fused form instead of the separated enzyme at the molecular level, we determined the crystal structure of HpMsrABC44S/C318S at 2.2 Å, which showed that a linker region (Hpiloop, 193–205) between two domains interacted with each HpMsrA or HpMsrB domain via three salt bridges (E193-K107, D197-R103, and K200-D339). Two acetate molecules in the active site pocket showed an sp2 planar electron density map in the crystal structure, which interacted with the conserved residues in fusion MsrABs from the pathogen. Biochemical and kinetic analyses revealed that Hpiloop is required to increase the catalytic efficiency of HpMsrAB. Two salt bridge mutants (D193A and E199A) were located at the entrance or tailgate of Hpiloop. Therefore, the linker region of the MsrAB fusion enzyme plays a key role in the structural stability and catalytic efficiency and provides a better understanding of why MsrAB exists in a fused form.

scholarly journals Increased Catalytic Efficiency following Gene Fusion of Bifunctional Methionine Sulfoxide Reductase Enzymes fromShewanella oneidensis†

Biochemistry ◽  
2007 ◽  
Vol 46 (49) ◽  
pp. 14153-14161 ◽  
Author(s):  
Baowei Chen ◽  
Lye Meng Markillie ◽  
Yijia Xiong ◽  
M. Uljana Mayer ◽  
Thomas C. Squier
Keyword(s):  
Gene Fusion ◽  
Catalytic Efficiency ◽  
Methionine Sulfoxide ◽  
Methionine Sulfoxide Reductase

scholarly journals Salt Bridges and Gating in the COOH-terminal Region of HCN2 and CNGA1 Channels

2004 ◽  
Vol 124 (6) ◽  
pp. 663-677 ◽  
Author(s):  
Kimberley B. Craven ◽  
William N. Zagotta
Keyword(s):  
Crystal Structure ◽  
Cyclic Nucleotide ◽  
Sequence Similarity ◽  
Terminal Region ◽  
Hcn Channels ◽  
Salt Bridges ◽  
Linker Region ◽  
Channel Opening ◽  
Intersubunit Interactions ◽  
Cnga1 Channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels and cyclic nucleotide-gated (CNG) channels are activated by the direct binding of cyclic nucleotides. The intracellular COOH-terminal regions exhibit high sequence similarity in all HCN and CNG channels. This region contains the cyclic nucleotide-binding domain (CNBD) and the C-linker region, which connects the CNBD to the pore. Recently, the structure of the HCN2 COOH-terminal region was solved and shown to contain intersubunit interactions between C-linker regions. To explore the role of these intersubunit interactions in intact channels, we studied two salt bridges in the C-linker region: an intersubunit interaction between C-linkers of neighboring subunits, and an intrasubunit interaction between the C-linker and its CNBD. We show that breaking these salt bridges in both HCN2 and CNGA1 channels through mutation causes an increase in the favorability of channel opening. The wild-type behavior of both HCN2 and CNGA1 channels is rescued by switching the position of the positive and negative residues, thus restoring the salt bridges. These results suggest that the salt bridges seen in the HCN2 COOH-terminal crystal structure are also present in the intact HCN2 channel. Furthermore, the similar effects of the mutations on HCN2 and CNGA1 channels suggest that these salt bridge interactions are also present in the intact CNGA1 channel. As disrupting the interactions leads to channels with more favorable opening transitions, the salt bridges appear to stabilize a closed conformation in both the HCN2 and CNGA1 channels. These results suggest that the HCN2 COOH-terminal crystal structure contains the C-linker regions in the resting configuration even though the CNBD is ligand bound, and channel opening involves a rearrangement of the C-linkers and, thus, disruption of the salt bridges. Discovering that one portion of the COOH terminus, the CNBD, can be in the activated configuration while the other portion, the C-linker, is not activated has lead us to suggest a novel modular gating scheme for HCN and CNG channels.

scholarly journals Noncatalytic Antioxidant Role forHelicobacter pyloriUrease

2018 ◽  
Vol 200 (17) ◽  
Author(s):  
Alan A. Schmalstig ◽  
Stéphane L. Benoit ◽  
Sandeep K. Misra ◽  
Joshua S. Sharp ◽  
Robert J. Maier
Keyword(s):  
Helicobacter Pylori ◽  
Methionine Sulfoxide ◽  
Methionine Sulfoxide Reductase ◽  
Repair System ◽  
Content Type ◽  
Urease Enzyme ◽  
Catalytic Role ◽  
Methionine Residues ◽  
H Pylori

ABSTRACTThe well-studied catalytic role of urease, the Ni-dependent conversion of urea into carbon dioxide and ammonia, has been shown to protectHelicobacter pyloriagainst the low pH environment of the stomach lumen. We hypothesized that the abundantly expressed urease protein can play another noncatalytic role in combating oxidative stress via Met residue-mediated quenching of harmful oxidants. Three catalytically inactive urease mutant strains were constructed by single substitutions of Ni binding residues. The mutant versions synthesize normal levels of urease, and the altered versions retained all methionine residues. The three site-directed urease mutants were able to better withstand a hypochlorous acid (HOCl) challenge than a ΔureABdeletion strain. The capacity of purified urease to protect whole cells via oxidant quenching was assessed by adding urease enzyme to nongrowing HOCl-exposed cells. No wild-type cells were recovered with oxidant alone, whereas urease addition significantly aided viability. These results suggest that urease can protectH. pyloriagainst oxidative damage and that the protective ability is distinct from the well-characterized catalytic role. To determine the capability of methionine sulfoxide reductase (Msr) to reduce oxidized Met residues in urease, purifiedH. pyloriurease was exposed to HOCl and a previously described Msr peptide repair mixture was added. Of the 25 methionine residues in urease, 11 were subject to both oxidation and to Msr-mediated repair, as identified by mass spectrometry (MS) analysis; therefore, the oxidant-quenchable Met pool comprising urease can be recycled by the Msr repair system. Noncatalytic urease appears to play an important role in oxidant protection.IMPORTANCEChronicHelicobacter pyloriinfection can lead to gastric ulcers and gastric cancers. The enzyme urease contributes to the survival of the bacterium in the harsh environment of the stomach by increasing the local pH. In addition to combating acid,H. pylorimust survive host-produced reactive oxygen species to persist in the gastric mucosa. We describe a cyclic amino acid-based antioxidant role of urease, whereby oxidized methionine residues can be recycled by methionine sulfoxide reductase to again quench oxidants. This work expands our understanding of the role of an already acknowledged pathogen virulence factor and specifically expands our knowledge ofH. pylorisurvival mechanisms.

scholarly journals Structure of Mycobacterium tuberculosis Methionine Sulfoxide Reductase A in Complex with Protein-Bound Methionine

2003 ◽  
Vol 185 (14) ◽  
pp. 4119-4126 ◽  
Author(s):  
Alexander B. Taylor ◽  
David M. Benglis, ◽  
Subramanian Dhandayuthapani ◽  
P. John Hart
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Pathogenic Bacteria ◽  
Methionine Sulfoxide ◽  
Methionine Sulfoxide Reductase ◽  
Host Cells ◽  
Cysteine Residues ◽  
Methionine Sulfoxide Reductase A ◽  
Methionine Residue ◽  
Reactive Nitrogen Intermediates ◽  
Methionine Residues

ABSTRACT Peptide methionine sulfoxide reductase (MsrA) repairs oxidative damage to methionine residues arising from reactive oxygen species and reactive nitrogen intermediates. MsrA activity is found in a wide variety of organisms, and it is implicated as one of the primary defenses against oxidative stress. Disruption of the gene encoding MsrA in several pathogenic bacteria responsible for infections in humans results in the loss of their ability to colonize host cells. Here, we present the X-ray crystal structure of MsrA from the pathogenic bacterium Mycobacterium tuberculosis refined to 1.5 Å resolution. In contrast to the three catalytic cysteine residues found in previously characterized MsrA structures, M. tuberculosis MsrA represents a class containing only two functional cysteine residues. The structure reveals a methionine residue of one MsrA molecule bound at the active site of a neighboring molecule in the crystal lattice and thus serves as an excellent model for protein-bound methionine sulfoxide recognition and repair.

scholarly journals Methionine Sulfoxide Reductase A (MsrA) Deficiency Affects the Survival of Mycobacterium smegmatis within Macrophages

2004 ◽  
Vol 186 (11) ◽  
pp. 3590-3598 ◽  
Author(s):  
T. Douglas ◽  
D. S. Daniel ◽  
B. K. Parida ◽  
C. Jagannath ◽  
S. Dhandayuthapani
Keyword(s):  
Mutant Strain ◽  
Mycobacterium Smegmatis ◽  
Type Strain ◽  
Pathogenic Bacteria ◽  
Wild Type Strain ◽  
Methionine Sulfoxide ◽  
Intracellular Survival ◽  
Methionine Sulfoxide Reductase ◽  
Wild Type ◽  
Methionine Sulfoxide Reductase A

ABSTRACT Methionine sulfoxide reductase A (MsrA) is an antioxidant repair enzyme which reduces oxidized methionine to methionine. Since oxidation of methionine in proteins impairs their function, an absence of MsrA leads to abnormalities in different organisms, including alterations in the adherence patterns and in vivo survival of certain pathogenic bacteria. To understand the role of MsrA in intracellular survival of bacteria, we disrupted the gene encoding MsrA in Mycobacterium smegmatis through homologous recombination. The msrA mutant strain of M. smegmatis exhibited significantly reduced intracellular survival in murine J774A.1 macrophages compared to the survival of its wild-type counterpart. Furthermore, immunofluorescence and immnunoblotting of phagosomes containing M. smegmatis strains revealed that the phagosomes with the msrA mutant strain acquired both p67phox of phagocyte NADPH oxidase and inducible nitric oxide synthase much earlier than the phagosomes with the wild-type strain. In addition, the msrA mutant strain of M. smegmatis was observed to be more sensitive to hydroperoxides than the wild-type strain was in vitro. These results suggest that MsrA plays an important role in both extracellular and intracellular survival of M. smegmatis.

scholarly journals GlycoTorch Vina: Improved Docking of Sulfated Sugars Using QM-derived Scoring Functions

2020 ◽  
Author(s):  
Eric Boittier ◽  
Jed Burns ◽  
Neha Gandhi ◽  
Vito Ferro
Keyword(s):  
Crystal Structure ◽  
Crystal Structures ◽  
Density Functional ◽  
Protein Complexes ◽  
Salt Bridge ◽  
Substantial Improvement ◽  
Standard Test ◽  
Scoring Functions ◽  
Salt Bridges ◽  
Gag Protein

Glycosaminoglycans (GAGs) are a family of anionic carbohydrates that play an essential role in the physiology and pathology of all eukaryotic life. Experimental determination of GAG-protein complexes remains difficult due to the considerable diversity in both carbohydrate linkage, and sulfation patterns. To complement existing methods of structural determination, we present our molecular docking tool, GlycoTorchVina (GTV), which demonstrates a substantial improvement at reproducing low energy conformations of GAGs compared with traditional docking programs. Based on the carbohydrate specific docking program VinaCarb (VC), GTV utilizes rotational energy functions, calculated using density functional theory (DFT), specifically designed for glycosidic linkages found in GAGs. The redocking accuracy of four programs (GTV, VC, AutoDock Vina and Glide) was tested over a set of 10 high-quality crystal structures containing co-crystallized GAGs (tetrasaccharides or longer). GTV outperformed other programs and was able to reproduce the native pose of eight structures and produced top-scoring docked poses that were on average only 1.8 Å RMSD away from the crystal structure. Although imitation of crystal structures is a standard test used for assessing the accuracy of docking programs, we illustrate how the underlying quality of the crystal structure, which is often overlooked during benchmarking, affects conclusions drawn from this approach. Statistical and theoretical investigations into charge-charge (“salt-bridge”) interactions are also presented. Again, DFT calculations were used to derive non-bonded potentials describing salt-bridges, and solvent-mediated charge-charge (“water-bridge”) interactions. These data suggest that water-bridges play an important, yet poorly understood, role in the structures of GAG-protein complexes.

Essential Role of the Linker Region in the Higher Catalytic Efficiency of a Bifunctional MsrA–MsrB Fusion Protein

Biochemistry ◽  
2016 ◽  
Vol 55 (36) ◽  
pp. 5117-5127 ◽  
Author(s):  
Ah-reum Han ◽  
Moon-Jung Kim ◽  
Geun-Hee Kwak ◽  
Jonghyeon Son ◽  
Kwang Yeon Hwang ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Essential Role ◽  
Catalytic Efficiency ◽  
Linker Region

scholarly journals Role of Helicobacter pylori methionine sulfoxide reductase in urease maturation

2013 ◽  
Vol 450 (1) ◽  
pp. 141-148 ◽  
Author(s):  
Lisa G. Kuhns ◽  
Manish Mahawar ◽  
Joshua S. Sharp ◽  
Stéphane Benoit ◽  
Robert J. Maier
Keyword(s):  
Helicobacter Pylori ◽  
Parent Strain ◽  
Specific Activity ◽  
Methionine Sulfoxide ◽  
Methionine Sulfoxide Reductase ◽  
Accessory Proteins ◽  
Wild Type ◽  
Methionine Residues ◽  
H Pylori

The persistence of the gastric pathogen Helicobacter pylori is due in part to urease and Msr (methionine sulfoxide reductase). Upon exposure to relatively mild (21% partial pressure of O2) oxidative stress, a Δmsr mutant showed both decreased urease specific activity in cell-free extracts and decreased nickel associated with the partially purified urease fraction as compared with the parent strain, yet urease apoprotein levels were the same for the Δmsr and wild-type extracts. Urease activity of the Δmsr mutant was not significantly different from the wild-type upon non-stress microaerobic incubation of strains. Urease maturation occurs through nickel mobilization via a suite of known accessory proteins, one being the GTPase UreG. Treatment of UreG with H2O2 resulted in oxidation of MS-identified methionine residues and loss of up to 70% of its GTPase activity. Incubation of pure H2O2-treated UreG with Msr led to reductive repair of nine methionine residues and recovery of up to full enzyme activity. Binding of Msr to both oxidized and non-oxidized UreG was observed by cross-linking. Therefore we conclude Msr aids the survival of H. pylori in part by ensuring continual UreG-mediated urease maturation under stress conditions.

scholarly journals Alkyl Hydroperoxide Reductase Repair by Helicobacter pylori Methionine Sulfoxide Reductase

2013 ◽  
Vol 195 (23) ◽  
pp. 5396-5401 ◽  
Author(s):  
S. L. Benoit ◽  
K. Bayyareddy ◽  
M. Mahawar ◽  
J. S. Sharp ◽  
R. J. Maier
Keyword(s):  
Helicobacter Pylori ◽  
Methionine Sulfoxide ◽  
Methionine Sulfoxide Reductase ◽  
Alkyl Hydroperoxide Reductase ◽  
Alkyl Hydroperoxide

scholarly journals Methionine Sulfoxide Reductase in Helicobacter pylori: Interaction with Methionine-Rich Proteins and Stress-Induced Expression

2006 ◽  
Vol 188 (16) ◽  
pp. 5839-5850 ◽  
Author(s):  
Praveen Alamuri ◽  
Robert J. Maier
Keyword(s):  
Helicobacter Pylori ◽  
Specific Activity ◽  
Methionine Sulfoxide ◽  
Stress Conditions ◽  
Methionine Sulfoxide Reductase ◽  
Iron Starvation ◽  
Protein Levels ◽  
Cell Extracts ◽  
Methionine Residues ◽  
H Pylori

ABSTRACT The reductive repair of oxidized methionine residues performed by methionine sulfoxide reductase is important for the gastric pathogen Helicobacter pylori to maintain persistent stomach colonization. Methionine-containing proteins that are targeted for repair by Msr were identified from whole-cell extracts (after cells were exposed to O2 stress) by using a coimmunoprecipitation approach. Proteins identified as Msr-interacting included catalase, GroEL, thioredoxin-1 (Trx1), and site-specific recombinase; with one exception (Trx1, the reductant for Msr) all these proteins have approximately twofold higher methionine (Met) content than other proteins. These Met-rich proteins were purified and were shown to individually form a cross-linked adduct with Msr. Catalase-specific activity in an msr strain was one-half that of the parent strain; this difference was only observed under oxidative stress conditions, and the activity was restored to nearly wild-type levels by adding Msr plus dithiothreitol to msr strain extracts. In agreement with the cross-linking study, pure Msr used Trx1 but not Trx2 as a reductant. Comparative structure modeling classified the H. pylori Msr in class II within the MsrB family, like the Neisseria enzymes. Pure H. pylori enzyme reduced only the R isomer of methyl p-tolyl-sulfoxide with an apparent Km of 4.1 mM for the substrate. Stress conditions (peroxide, peroxynitrite, and iron starvation) all caused approximately 3- to 3.5-fold transcriptional up-regulation of msr. Neither the O2 level during growth nor the use of background regulatory mutants had a significant effect on msr transcription. Late log and stationary phase cultures had the highest Msr protein levels and specific activity.

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