scholarly journals Burst Kinetics and Redox Transformations of the Active Site Manganese Ion in Oxalate Oxidase

2007 ◽  
Vol 282 (10) ◽  
pp. 7011-7023 ◽  
Author(s):  
Mei M. Whittaker ◽  
Heng-Yen Pan ◽  
Erik T. Yukl ◽  
James W. Whittaker
Keyword(s):  
Active Site ◽  
Oxalate Oxidase ◽  
Redox Transformations ◽  
Manganese Ion ◽  
Burst Kinetics

scholarly journals A comparative study of class-D β-lactamases

1993 ◽  
Vol 292 (2) ◽  
pp. 555-562 ◽  
Author(s):  
P Ledent ◽  
X Raquet ◽  
B Joris ◽  
J Van Beeumen ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Gene Sequence ◽  
Catalytic Properties ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Beta Lactamase ◽  
Serine Residue ◽  
Beta Lactamases ◽  
Class D ◽  
Burst Kinetics

Three class-D beta-lactamases (OXA2, OXA1 and PSE2) were produced and purified to protein homogeneity. 6 beta-Iodopenicillanate inactivated the OXA2 enzyme without detectable turnover. Labelling of the same beta-lactamase with 6 beta-iodo[3H]penicillanate allowed the identification of Ser-70 as the active-site serine residue. In agreement with previous reports, the apparent M(r) of the OXA2 enzyme as determined by molecular-sieve filtration, was significantly higher than that deduced from the gene sequence, but this was not due to an equilibrium between a monomer and a dimer. The heterogeneity of the OXA2 beta-lactamase on ion-exchange chromatography contrasted with the similarity of the catalytic properties of the various forms. A first overview of the enzymic properties of the three ‘oxacillinases’ is presented. With the OXA2 enzyme, ‘burst’ kinetics, implying branched pathways, seemed to prevail with many substrates.

scholarly journals Barley (Hordeum vulgare) oxalate oxidase is a manganese-containing enzyme

1999 ◽  
Vol 343 (1) ◽  
pp. 185-190 ◽  
Author(s):  
Laura REQUENA ◽  
Stephen BORNEMANN
Keyword(s):  
Hordeum Vulgare ◽  
Epr Spectroscopy ◽  
Active Site ◽  
Homology Model ◽  
Direct Conversion ◽  
Oxalate Oxidase ◽  
Metal Analysis ◽  
Seedling Root

Oxalate oxidase (EC 1.2.3.4) catalyses the conversion of oxalate and dioxygen into CO2 and H2O2. The barley (Hordeum vulgare) seedling root enzyme was purified to homogeneity and shown by metal analysis and EPR spectroscopy to contain Mn(II) at up to 0.80 atom per subunit. The involvement of Mn and neither flavin, Cu nor Fe in the direct conversion of dioxygen to H2O2 makes oxalate oxidase unique. A model of the active site of the holoenzyme based on a homology model of the apoenzyme is proposed.

scholarly journals Analysis of the HindIII-catalyzed reaction by time-resolved crystallography

2015 ◽  
Vol 71 (2) ◽  
pp. 256-265 ◽  
Author(s):  
Takashi Kawamura ◽  
Tomoki Kobayashi ◽  
Nobuhisa Watanabe
Keyword(s):  
Electron Density ◽  
Active Site ◽  
Dna Cleavage ◽  
Metal Ion ◽  
Manganese Ions ◽  
Metal Ion Binding ◽  
Time Resolved ◽  
Manganese Ion ◽  
Ion Binding Sites ◽  
Metal Ion Binding Sites

In order to investigate the mechanism of the reaction catalyzed by HindIII, structures of HindIII–DNA complexes with varying durations of soaking time in cryoprotectant buffer containing manganese ions were determined by the freeze-trap method. In the crystal structures of the complexes obtained after soaking for a longer duration, two manganese ions, indicated by relatively higher electron density, are clearly observed at the two metal ion-binding sites in the active site of HindIII. The increase in the electron density of the two metal-ion peaks followed distinct pathways with increasing soaking times, suggesting variation in the binding rate constant for the two metal sites. DNA cleavage is observed when the second manganese ion appears, suggesting that HindIII uses the two-metal-ion mechanism, or alternatively that its reactivity is enhanced by the binding of the second metal ion. In addition, conformational change in a loop near the active site accompanies the catalytic reaction.

scholarly journals The identity of the active site of oxalate decarboxylase and the importance of the stability of active-site lid conformations1

2007 ◽  
Vol 407 (3) ◽  
pp. 397-406 ◽  
Author(s):  
Victoria J. Just ◽  
Matthew R. Burrell ◽  
Laura Bowater ◽  
Iain McRobbie ◽  
Clare E. M. Stevenson ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Structural Information ◽  
Side Reaction ◽  
Oxalate Oxidase ◽  
Decarboxylase Activity ◽  
Oxalate Decarboxylase ◽  
Catalytically Active ◽  
The Stability ◽  
Kinetics Of

Oxalate decarboxylase (EC 4.1.1.2) catalyses the conversion of oxalate into carbon dioxide and formate. It requires manganese and, uniquely, dioxygen for catalysis. It forms a homohexamer and each subunit contains two similar, but distinct, manganese sites termed sites 1 and 2. There is kinetic evidence that only site 1 is catalytically active and that site 2 is purely structural. However, the kinetics of enzymes with mutations in site 2 are often ambiguous and all mutant kinetics have been interpreted without structural information. Nine new site-directed mutants have been generated and four mutant crystal structures have now been solved. Most mutants targeted (i) the flexibility (T165P), (ii) favoured conformation (S161A, S164A, D297A or H299A) or (iii) presence (Δ162–163 or Δ162–164) of a lid associated with site 1. The kinetics of these mutants were consistent with only site 1 being catalytically active. This was particularly striking with D297A and H299A because they disrupted hydrogen bonds between the lid and a neighbouring subunit only when in the open conformation and were distant from site 2. These observations also provided the first evidence that the flexibility and stability of lid conformations are important in catalysis. The deletion of the lid to mimic the plant oxalate oxidase led to a loss of decarboxylase activity, but only a slight elevation in the oxalate oxidase side reaction, implying other changes are required to afford a reaction specificity switch. The four mutant crystal structures (R92A, E162A, Δ162–163 and S161A) strongly support the hypothesis that site 2 is purely structural.

Oxalate Decarboxylase and Oxalate Oxidase Activities Can Be Interchanged with a Specificity Switch of up to 282 000 by Mutating an Active Site Lid†,‡

Biochemistry ◽  
2007 ◽  
Vol 46 (43) ◽  
pp. 12327-12336 ◽  
Author(s):  
Matthew R. Burrell ◽  
Victoria J. Just ◽  
Laura Bowater ◽  
Shirley A. Fairhurst ◽  
Laura Requena ◽  
...  
Keyword(s):  
Active Site ◽  
Oxalate Oxidase ◽  
Oxalate Decarboxylase

scholarly journals Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants

2013 ◽  
Vol 27 (S1) ◽  
Author(s):  
Ellen W Moomaw ◽  
Eric Hoffer ◽  
Patricia Moussatche ◽  
John Salerno ◽  
Morgan Grant ◽  
...  
Keyword(s):  
Active Site ◽  
Spectroscopic Studies ◽  
Oxalate Oxidase ◽  
Putative Active Site ◽  
Active Site Mutants

scholarly journals Reaction of Peroxynitrite with Mn-Superoxide Dismutase

2001 ◽  
Vol 276 (15) ◽  
pp. 11631-11638 ◽  
Author(s):  
Celia Quijano ◽  
Daniel Hernandez-Saavedra ◽  
Laura Castro ◽  
Joe M. McCord ◽  
Bruce A. Freeman ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Rate Constant ◽  
Active Site ◽  
Manganese Superoxide Dismutase ◽  
Order Rate Constant ◽  
Metal Centers ◽  
Manganese Ion ◽  
Hydroxyphenylacetic Acid

Manganese superoxide dismutase (Mn-SOD), a critical mitochondrial antioxidant enzyme, becomes inactivated and nitratedin vitroand potentiallyin vivoby peroxynitrite. Since peroxynitrite readily reacts with transition metal centers, we assessed the role of the manganese ion in the reaction between peroxynitrite and Mn-SOD. Peroxynitrite reacts with human recombinant andEscherichia coliMn-SOD with a second order rate constant of 1.0 ± 0.2 × 105and 1.4 ± 0.2 × 105m−1s−1at pH 7.47 and 37 °C, respectively. TheE. coliapoenzyme, obtained by removing the manganese ion from the active site, presents a rate constant <104m−1s−1for the reaction with peroxynitrite, whereas that of the manganese-reconstituted apoenzyme (apo/Mn) was comparable to that of the holoenzyme. Peroxynitrite-dependent nitration of 4-hydroxyphenylacetic acid was increased 21% by Mn-SOD. The apo/Mn also promoted nitration, but the apo and the zinc-substituted apoenzyme (apo/Zn) enzymes did not. The extent of tyrosine nitration in the enzyme was also affected by the presence and nature (i.e.manganese or zinc) of the metal center in the active site. For comparative purposes, we also studied the reaction of peroxynitrite with low molecular weight complexes of manganese and zinc with tetrakis-(4-benzoic acid) porphyrin (tbap). Mn(tbap) reacts with peroxynitrite with a rate constant of 6.8 ± 0.1 × 104m−1s−1and maximally increases nitration yields by 350%. Zn(tbap), on the other hand, affords protection against nitration. Our results indicate that the manganese ion in Mn-SOD plays an important role in the decomposition kinetics of peroxynitrite and in peroxynitrite-dependent nitration of self and remote tyrosine residues.

scholarly journals Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants

PLoS ONE ◽  
2013 ◽  
Vol 8 (3) ◽  
pp. e57933 ◽  
Author(s):  
Ellen W. Moomaw ◽  
Eric Hoffer ◽  
Patricia Moussatche ◽  
John C. Salerno ◽  
Morgan Grant ◽  
...  
Keyword(s):  
Active Site ◽  
Spectroscopic Studies ◽  
Oxalate Oxidase ◽  
Putative Active Site ◽  
Active Site Mutants

scholarly journals LanD-like Flavoprotein-Catalyzed Aminovinyl-Cysteine Formation through Oxidative Decarboxylation and Cyclization of a Peptide at the C-Terminus

2020 ◽  
Author(s):  
Jingyu Liu ◽  
Yanping Qiu ◽  
Tao Fu ◽  
Miao Li ◽  
Yuqing Li ◽  
...  
Keyword(s):  
Active Site ◽  
Proximity Effect ◽  
Structural Diversity ◽  
Oxidative Decarboxylation ◽  
Peptide Substrate ◽  
Post Translational Modifications ◽  
Peptide Cyclization ◽  
Redox Transformations ◽  
Peptide Formation ◽  
C Terminus

ABSTRACTAminovinyl-cysteine residues arise from processing the C-terminal l-Cys and an internal l-Ser/l-Thr or l-Cys of a peptide. Formation of these nonproteinogenic amino acids, which occur in a macrocyclic ring of diverse ribosomally synthesized lanthipeptides and non-lanthipeptides, remains poorly understood. Here, we report that LanD-like flavoproteins in the biosynthesis of distinct non-lanthipeptides share an unexpected dual activity for aminovinyl-cysteine formation. Each flavoprotein catalyzes oxidative decarboxylation of the C-terminal l-Cys and couples the resulting enethiol nucleophile with the internal residue to afford a thioether linkage for peptide cyclization. The cyclization step, which largely depends on proximity effect by positioning the enethiol intermediate with a bent conformation at the active site, can be substrate-dependent, proceeding inefficiently through nucleophilic substitution for an unmodified peptide or efficiently through Michael addition for a dehydrated/dethiolated peptide. Uncovering this unusual flavin-dependent paradigm for thioether residue formation advances the understanding in the biosynthesis of aminovinyl-cysteine-containing RiPPs and renews interest in flavoproteins, particularly those involved in non-redox transformations. LanD-like flavoproteins activity, which is flexible in peptide substrate and amenable for evolution by engineering, can be combined with different post-translational modifications for structural diversity, thereby holding promise for peptide macrocyclization/functionalization in drug development by chemoenzymatic or synthetic biology approaches.

Modeling based on the structure of vicilins predicts a histidine cluster in the active site of oxalate oxidase

1998 ◽  
Vol 46 (4) ◽  
pp. 488-493 ◽  
Author(s):  
Paul J. Gane ◽  
Jim M. Dunwell ◽  
Jim Warwickr
Keyword(s):  
Active Site ◽  
Oxalate Oxidase
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