Identification of tunnels in proteins, nucleic acids, inorganic materials and molecular ensembles

The catalytic mechanism of nitrate reduction by periplasmic nitrate reductases has been investigated using theoretical and computational means. We have found that the nitrate molecule binds to the active site with the Mo ion in the +6 oxidation state. Electron transfer to the active site occurs only in the proton-electron transfer stage, where the Mo(V) species plays an important role in catalysis. The presence of the sulfur atom in the molybdenum coordination sphere creates a pseudo-dithiolene ligand that protects it from any direct attack from the solvent. Upon the nitrate binding there is a conformational rearrangement of this ring that allows the direct contact of the nitrate with Mo(VI) ion. This rearrangement is stabilized by the conserved methionines Met141 and Met308. The reduction of nitrate into nitrite occurs in the second step of the mechanism where the two dimethyl-dithiolene ligands have a key role in spreading the excess of negative charge near the Mo atom to make it available for the chemical reaction. The reaction involves the oxidation of the sulfur atoms and not of the molybdenum as previously suggested. The mechanism involves a molybdenum and sulfur-based redox chemistry instead of the currently accepted redox chemistry based only on the Mo ion. The second part of the mechanism involves two protonation steps that are promoted by the presence of Mo(V) species. Mo(VI) intermediates might also be present in this stage depending on the availability of protons and electrons. Once the water molecule is generated only the Mo(VI) species allow water molecule dissociation, and, the concomitant enzymatic turnover.

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“…Using the Caver algorithm 25,26 we have also found two additional channels that also point towards the molybdenum center (see Fig. 3).…”

Section: Structural Information Of the Nitrate Reductase Active Sitementioning

confidence: 93%

The effect of the sixth sulfur ligand in the catalytic mechanism of periplasmic nitrate reductase

et al. 2009

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“…Tunnels in Static Structures-The tunnels connecting the active site with bulk solvent in LinB WT and LinB L177W were analyzed using the CAVER program (15,17,42), which was developed specifically for identification and visualization of tunnels in proteins with buried active sites. The current version …”

Section: Kinetics Of Bromide Ionmentioning

confidence: 99%

A Single Mutation in a Tunnel to the Active Site Changes the Mechanism and Kinetics of Product Release in Haloalkane Dehalogenase LinB

Biedermannová

Prokop

Góra

et al. 2012

Journal of Biological Chemistry

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Background: Tunnel properties affect ligand passage in enzymes with buried active sites. Results: A tunnel mutation from leucine to tryptophan changes the mechanism of bromide ion release from haloalkane dehalogenase LinB. Conclusion:Interactions of the bromide ion with the tryptophan increase free energy barrier for its passage, causing the reaction mechanism change. Significance: The results provide guidelines for enzyme engineering.

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“…The cavity volume of all 4 monomers was measured along each trajectory using the software suite CAVER. 29 Additionally, the compactness of the tetramer was calculated for each snapshot of the trajectory using the method described in the context of the GA. 24 Creation and purification of L-ASN recombinants L-ASN recombinants were created as described in Patel et al 23 Preparation of purified recombinant proteins and estimation of protein molecular weight and aggregation by SEC-MALLS (size exclusion chromatography coupled with multi-angle laser light scattering) were carried out by the Protein Production Division of the Technology Facility in the Department of Biology at the University of York, United Kingdom.…”

Section: Analysis Of MD Trajectoriesmentioning

confidence: 99%

Rational engineering of L-asparaginase reveals importance of dual activity for cancer cell toxicity

Offman

Król

Patel

et al. 2011

Blood

134

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Using proteins in a therapeutic context often requires engineering to modify functionality and enhance efficacy. We have previously reported that the therapeutic antileukemic protein macromolecule Escherichia coli L-asparaginase is degraded by leukemic lysosomal cysteine proteases. In the present study, we successfully engineered L-asparaginase to resist proteolytic cleavage and at the same time improve activity. We employed a novel combination of mutant sampling using a genetic algorithm in tandem with flexibility studies using molecular dynamics to investigate the impact of lid-loop and mutations on drug activity. Applying these methods, we successfully predicted the more active L-asparaginase mutants N24T and N24A. For the latter, a unique hydrogen bond network contributes to higher activity. Furthermore, interface mutations controlling secondary glutaminase activity demonstrated the importance of this enzymatic activity for drug cytotoxicity. All selected mutants were expressed, purified, and tested for activity and for their ability to form the active tetrameric form. By introducing the N24A and N24A R195S mutations to the drug L-asparaginase, we are a step closer to individualized drug design. (Blood. 2011; 117(5):1614-1621)

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Identification of tunnels in proteins, nucleic acids, inorganic materials and molecular ensembles

Cited by 54 publications

References 60 publications

The effect of the sixth sulfur ligand in the catalytic mechanism of periplasmic nitrate reductase

The effect of the sixth sulfur ligand in the catalytic mechanism of periplasmic nitrate reductase

A Single Mutation in a Tunnel to the Active Site Changes the Mechanism and Kinetics of Product Release in Haloalkane Dehalogenase LinB

Rational engineering of L-asparaginase reveals importance of dual activity for cancer cell toxicity

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