Insights into Redox Partner Interactions and Substrate Binding in Nitrite Reductase fromAlcaligenes xylosoxidans: Crystal Structures of the Trp138His and His313Gln Mutants†,‡

Biochemistry ◽  
2004 ◽  
Vol 43 (51) ◽  
pp. 16311-16319 ◽  
Author(s):  
Mark L. Barrett ◽  
Roger L. Harris ◽  
Svetlana Antonyuk ◽  
Michael A. Hough ◽  
Mark J. Ellis ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Nitrite Reductase ◽  
Substrate Binding ◽  
Redox Partner

scholarly journals SERR Spectroelectrochemical Study of Cytochrome cd1 Nitrite Reductase Co-Immobilized with Physiological Redox Partner Cytochrome c552 on Biocompatible Metal Electrodes

PLoS ONE ◽  
2015 ◽  
Vol 10 (6) ◽  
pp. e0129940 ◽  
Author(s):  
Célia M. Silveira ◽  
Pedro O. Quintas ◽  
Isabel Moura ◽  
José J. G. Moura ◽  
Peter Hildebrandt ◽  
...  
Keyword(s):  
Nitrite Reductase ◽  
Metal Electrodes ◽  
Redox Partner ◽  
Cytochrome C552 ◽  
Cytochrome Cd1

scholarly journals Domain sliding of two Staphylococcus aureus N-acetylglucosaminidases enables their substrate-binding prior to its catalysis

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Sara Pintar ◽  
Jure Borišek ◽  
Aleksandra Usenik ◽  
Andrej Perdih ◽  
Dušan Turk
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Drug Targets ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Human Pathogen ◽  
Active Site Cleft ◽  
Novel Drug ◽  
Novel Drug Targets

AbstractTo achieve productive binding, enzymes and substrates must align their geometries to complement each other along an entire substrate binding site, which may require enzyme flexibility. In pursuit of novel drug targets for the human pathogen S. aureus, we studied peptidoglycan N-acetylglucosaminidases, whose structures are composed of two domains forming a V-shaped active site cleft. Combined insights from crystal structures supported by site-directed mutagenesis, modeling, and molecular dynamics enabled us to elucidate the substrate binding mechanism of SagB and AtlA-gl. This mechanism requires domain sliding from the open form observed in their crystal structures, leading to polysaccharide substrate binding in the closed form, which can enzymatically process the bound substrate. We suggest that these two hydrolases must exhibit unusual extents of flexibility to cleave the rigid structure of a bacterial cell wall.

Molecular interactions between multihaem cytochromes: probing the protein–protein interactions between pentahaem cytochromes of a nitrite reductase complex

2011 ◽  
Vol 39 (1) ◽  
pp. 263-268 ◽  
Author(s):  
Colin Lockwood ◽  
Julea N. Butt ◽  
Thomas A. Clarke ◽  
David J. Richardson
Keyword(s):  
Protein Interactions ◽  
Nitrite Reductase ◽  
Analytical Ultracentrifugation ◽  
Gel Filtration ◽  
Protein Protein Interactions ◽  
Physiological Conditions ◽  
Sulfate Reducing ◽  
Redox Partner ◽  
Electron Reduction ◽  
Reducing Bacteria

The cytochrome c nitrite reductase NrfA is a 53 kDa pentahaem enzyme that crystallizes as a decahaem homodimer. NrfA catalyses the reduction of NO2− to NH4+ through a six electron reduction pathway that is of major physiological significance to the anaerobic metabolism of enteric and sulfate reducing bacteria. NrfA receives electrons from the 21 kDa pentahaem NrfB donor protein. This requires that redox complexes form between the NrfA and NrfB pentahaem cytochromes. The formation of these complexes can be monitored using a range of methodologies for studying protein–protein interactions, including dynamic light scattering, gel filtration, analytical ultracentrifugation and visible spectroscopy. These methods have been used to show that oxidized NrfA exists in dynamic monomer–dimer equilibrium with a Kd (dissociation constant) of 4 μM. Significantly, the monomeric and dimeric forms of NrfA are equally active for either the six electron reduction of NO2− or HSO3−. When mixed together, NrfA and NrfB exist in equilibrium with NrfAB, which is described by a Kd of 50 nM. Thus, since NrfA and NrfB are present in micromolar concentrations in the periplasmic compartment, it is likely that NrfB remains tightly associated with its NrfA redox partner under physiological conditions.

The nature of the substrate binding site in copper nitrite reductase from Achromobacter xylosoxidans (AxNiR)

1995 ◽  
Vol 59 (2-3) ◽  
pp. 711
Author(s):  
R.W. Strange ◽  
J.G. Grossmann ◽  
F.E. Dodd ◽  
S.S. Hasnain ◽  
Z.H.L. Abraham ◽  
...  
Keyword(s):  
Binding Site ◽  
Nitrite Reductase ◽  
Substrate Binding ◽  
Achromobacter Xylosoxidans ◽  
Substrate Binding Site

Substrate Binding to a Nitrite Reductase Induces a Spin Transition

2010 ◽  
Vol 114 (16) ◽  
pp. 5563-5566 ◽  
Author(s):  
Gabriel Martins ◽  
Luisa Rodrigues ◽  
Filipa M. Cunha ◽  
Daniela Matos ◽  
Peter Hildebrandt ◽  
...  
Keyword(s):  
Nitrite Reductase ◽  
Substrate Binding ◽  
Spin Transition

scholarly journals Molecular recognition of sugar binding in a melibiose transporter MelB by X-ray crystallography

2020 ◽  
Author(s):  
Lan Guan ◽  
Parameswaran Hariharan
Keyword(s):  
Molecular Recognition ◽  
Binding Site ◽  
Crystal Structures ◽  
Mammalian Cells ◽  
Substrate Binding ◽  
Structural Basis ◽  
Cooperative Binding ◽  
Recognition Mechanism ◽  
Substrate Binding Site ◽  
X Ray

AbstractThe symporter melibiose permease MelB is the best-studied representative from MFS_2 family and the only protein in this large family with crystal structure determined. Previous thermodynamic studies show that MelB utilizes a cooperative binding as the core mechanism for its obligatory symport. Here we present two sugar-bound X-ray crystal structures of a Salmonella typhimurium MelB D59C uniport mutant that binds and catalyzes melibiose transport uncoupled to either cation, as determined by biochemical and biophysical characterizations. The two structures with bound nitrophenyl-α-D-galactoside or dodecyl-β-D-melibioside, which were refined to a resolution of 3.05 or 3.15 Å, respectively, are virtually identical at an outward-facing conformation; each one contains a α-galactoside molecule in the middle of protein. In the substrate-binding site, the galactosyl moiety on both ligands are at an essentially same configuration, so a galactoside specificity determinant pocket can be recognized, and hence the molecular recognition mechanism for the binding of sugar in MelB is deciphered. The data also allow to assign the conserved cation-binding pocket, which is directly connected to the sugar specificity determinant pocket. The intimate connection between the two selection sites lays the structural basis for the cooperative binding and coupled transport. This key structural finding answered the long-standing question on the substrate binding for the Na+-coupled MFS family of transporters.SignificanceMajor facilitator superfamily_2 transporters contain >10,000 members that are widely expressed from bacteria to mammalian cells, and catalyze uptake of varied nutrients from sugars to phospholipids. While several crystal structures with bound sugar for other MFS permeases have been determined, they are either uniporters or symporters coupled solely to H+. MelB catalyzes melibiose symport with either Na+, Li+, or H+, a prototype for Na+-coupled MFS transporters, but its sugar recognition has been a long-unsolved puzzle. Two high-resolution crystal structures presented here clearly reveal the molecular recognition mechanism for the binding of sugar in MelB. The substrate-binding site is characterized with a small specificity groove adjoining a large nonspecific cavity, which could offer a potential for future exploration of active transporters for drug delivery.

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