scholarly journals Biochemical and structural characterization ofKlebsiella pneumoniaeoxamate amidohydrolase in the uric acid degradation pathway

2016 ◽  
Vol 72 (6) ◽  
pp. 808-816 ◽  
Author(s):  
Katherine A. Hicks ◽  
Steven E. Ealick
Keyword(s):  
Uric Acid ◽  
Active Site ◽  
Degradation Pathway ◽  
Α Subunit ◽  
Acid Degradation ◽  
Substrate Complex ◽  
Steady State Kinetics ◽  
Biochemical Studies ◽  
Kinetics Of ◽  
Insight Into

HpxW from the ubiquitous pathogenKlebsiella pneumoniaeis involved in a novel uric acid degradation pathway downstream from the formation of oxalurate. Specifically, HpxW is an oxamate amidohydrolase which catalyzes the conversion of oxamate to oxalate and is a member of the Ntn-hydrolase superfamily. HpxW is autoprocessed from an inactive precursor to form a heterodimer, resulting in a 35.5 kDa α subunit and a 20 kDa β subunit. Here, the structure of HpxW is presented and the substrate complex is modeled. In addition, the steady-state kinetics of this enzyme and two active-site variants were characterized. These structural and biochemical studies provide further insight into this class of enzymes and allow a mechanism for catalysis consistent with other members of the Ntn-hydrolase superfamily to be proposed.

scholarly journals Selection for Growth on 3-Nitrotoluene by 2-Nitrotoluene-Utilizing Acidovorax sp. Strain JS42 Identifies Nitroarene Dioxygenases with Altered Specificities

2014 ◽  
Vol 81 (1) ◽  
pp. 309-319 ◽  
Author(s):  
Kristina M. Mahan ◽  
Joseph T. Penrod ◽  
Kou-San Ju ◽  
Natascia Al Kass ◽  
Watumesa A. Tan ◽  
...  
Keyword(s):  
Active Site ◽  
Sole Source ◽  
Degradation Pathway ◽  
Short Term ◽  
Gene Encoding ◽  
Α Subunit ◽  
Content Type ◽  
Term Selection ◽  
Genes Encoding ◽  
Related Enzyme

ABSTRACTAcidovoraxsp. strain JS42 uses 2-nitrotoluene as a sole source of carbon and energy. The first enzyme of the degradation pathway, 2-nitrotoluene 2,3-dioxygenase, adds both atoms of molecular oxygen to 2-nitrotoluene, forming nitrite and 3-methylcatechol. All three mononitrotoluene isomers serve as substrates for 2-nitrotoluene dioxygenase, but strain JS42 is unable to grow on 3- or 4-nitrotoluene. Using both long- and short-term selections, we obtained spontaneous mutants of strain JS42 that grew on 3-nitrotoluene. All of the strains obtained by short-term selection had mutations in the gene encoding the α subunit of 2-nitrotoluene dioxygenase that changed isoleucine 204 at the active site to valine. Those strains obtained by long-term selections had mutations that changed the same residue to valine, alanine, or threonine or changed the alanine at position 405, which is just outside the active site, to glycine. All of these changes altered the regiospecificity of the enzymes with 3-nitrotoluene such that 4-methylcatechol was the primary product rather than 3-methylcatechol. Kinetic analyses indicated that the evolved enzymes had enhanced affinities for 3-nitrotoluene and were more catalytically efficient with 3-nitrotoluene than the wild-type enzyme. In contrast, the corresponding amino acid substitutions in the closely related enzyme nitrobenzene 1,2-dioxygenase were detrimental to enzyme activity. When cloned genes encoding the evolved dioxygenases were introduced into a JS42 mutant lacking a functional dioxygenase, the strains acquired the ability to grow on 3-nitrotoluene but with significantly longer doubling times than the evolved strains, suggesting that additional beneficial mutations occurred elsewhere in the genome.

scholarly journals Characterization of the Complete Uric Acid Degradation Pathway in the Fungal Pathogen Cryptococcus neoformans

PLoS ONE ◽  
2013 ◽  
Vol 8 (5) ◽  
pp. e64292 ◽  
Author(s):  
I. Russel Lee ◽  
Liting Yang ◽  
Gaseene Sebetso ◽  
Rebecca Allen ◽  
Thi H. N. Doan ◽  
...  
Keyword(s):  
Uric Acid ◽  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Degradation Pathway ◽  
Acid Degradation

scholarly journals Molecular basis for catabolism of the abundant metabolite trans-4-hydroxy-L-proline by a microbial glycyl radical enzyme

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Lindsey RF Backman ◽  
Yolanda Y Huang ◽  
Mary C Andorfer ◽  
Brian Gold ◽  
Ronald T Raines ◽  
...  
Keyword(s):  
Active Site ◽  
Metabolic Pathways ◽  
Molecular Basis ◽  
Radical Mechanism ◽  
Human Gut ◽  
Clostridioides Difficile ◽  
Glycyl Radical ◽  
Biochemical Studies ◽  
Glycyl Radical Enzyme ◽  
Insight Into

The glycyl radical enzyme (GRE) superfamily utilizes a glycyl radical cofactor to catalyze difficult chemical reactions in a variety of anaerobic microbial metabolic pathways. Recently, a GRE, trans-4-hydroxy-L-proline (Hyp) dehydratase (HypD), was discovered that catalyzes the dehydration of Hyp to (S)-Δ1-pyrroline-5-carboxylic acid (P5C). This enzyme is abundant in the human gut microbiome and also present in prominent bacterial pathogens. However, we lack an understanding of how HypD performs its unusual chemistry. Here, we have solved the crystal structure of HypD from the pathogen Clostridioides difficile with Hyp bound in the active site. Biochemical studies have led to the identification of key catalytic residues and have provided insight into the radical mechanism of Hyp dehydration.

ВЛИЯНИЕ ОСМОЛИТОВ НА БИОЛЮМИНЕСЦЕНТНУЮ РЕАКЦИЮ БАКТЕРИЙ: СТРУКТУРНО-ДИНАМИЧЕСКИЕ АСПЕКТЫ

2020 ◽  
Vol 65 (6) ◽  
pp. 1135-1141
Author(s):  
Л.А. Суковатый ◽  
◽  
А.Е. Лисица ◽  
В.А. Кратасюк ◽  
Е.В. Немцева ◽  
...  
Keyword(s):  
Quantum Yield ◽  
Active Site ◽  
Dynamics Simulation ◽  
Water Molecules ◽  
Bacterial Luciferase ◽  
Α Subunit ◽  
The Media ◽  
Viscous Media ◽  
Kinetics Of ◽  
Yield Decrease

The effects of viscous media with glycerol and sucrose (10-40%) on the kinetics of the bacterial bioluminescent reaction have been investigated by stopped-flow technique. Increment of quantum yield in media with 10% of both osmolytes was shown. Higher concentrations of glycerol, up to 30-40%, were found to reduce the efficiency of the reaction, while this effect was not observed in the media with sucrose. The molecular dynamics simulation was used to study the structure of bacterial luciferase surrounded by either water molecules solely or by mixture of water with various numbers of glycerol/sucrose molecules. It was found that both cosolvents at studied concentrations did not cause a significant change in conformation of bacterial luciferase. The calculated root mean square fluctuations for Cα-atoms of bacterial luciferase α-subunit indicated that the higher flexibility of the enzyme mobile loop could be responsible for increment of quantum yield in the presence of 10% of both osmolytes. The active site of bacterial luciferase was found to be accessible for glycerol molecules while sucrose did not enter catalytic gorge. Moreover, at 30% and 40% concentration the glycerol molecules were found to locate in the active site of bacterial luciferase throughout the whole simulation time (40 ns) and to exclude water molecules, which can explain the experimentally obtained reaction quantum yield decrease.

scholarly journals The effects of hydrogen ion concentration on the simplest steady-state enzyme systems

1971 ◽  
Vol 123 (3) ◽  
pp. 445-453 ◽  
Author(s):  
P. Ottolenghi
Keyword(s):  
Steady State ◽  
Active Site ◽  
Hydrogen Ion ◽  
Ion Concentration ◽  
Hydrogen Ion Concentration ◽  
Substrate Complex ◽  
Perturbation Term ◽  
Enzyme Substrate ◽  
Enzyme Systems ◽  
Steady State Kinetics

Laidler (1955) showed that consideration of the effect of pH on enzymic mechanisms that obey steady-state kinetics leads to the inclusion in the equations of a ‘perturbation term’ that can introduce curvature into the Lineweaver–Burk plots. He also stated conditions in which this term vanishes. This term can lead to apparent activation by substrate. Further, several cases are shown in which simplification, but not disappearance, of the perturbation term can lead to linearity of Lineweaver–Burk plots. These cases arise when the ionization of groups at the active site either is unaffected or is completely prevented when the enzyme–substrate complex is formed. It is also shown that V(app.) can vary with pH without a concomitant change in Km(app.) in certain cases that obey steady-state kinetics without implying that Km=Ks. When the perturbation term is significant, Dixon's (1953) rules for the calculation of pK values will not always apply.

scholarly journals Electrogenic Sodium–Sodium Exchange Carried Out by Na,k -Atpase Containing the Amino Acid Substitution Glu779ala

2000 ◽  
Vol 116 (1) ◽  
pp. 61-74 ◽  
Author(s):  
R. Daniel Peluffo ◽  
José M. Argüello ◽  
Jerry B Lingrel ◽  
Joshua R. Berlin
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
High Capacity ◽  
Outward Current ◽  
Α Subunit ◽  
High Affinity ◽  
High Level ◽  
Kinetics Of ◽  
Insight Into

Na,K -ATPase containing the amino acid substitution glutamate to alanine at position 779 of the α subunit (Glu779Ala) supports a high level of Na-ATPase and electrogenic Na+–Na+ exchange activityin the absence of K +. In microsomal preparations of Glu779Ala enzyme, the Na+ concentration for half maximal activation of Na-ATPase activity was 161 ± 14 mM (n = 3). Furthermore, enzyme activity with 800 mM Na+ was found to be similar in the presence and absence of 20 mM K +. These results showed that Na+, with low affinity, could stimulate enzyme turnover as effectively as K +. To gain further insight into the mechanism of this enzyme activity, HeLa cells expressing Glu779Ala enzyme were voltage clamped with patch electrodes containing 115 mM Na+ during superfusion in K +-free solutions. Electrogenic Na+–Na+ exchange was observed as an ouabain-inhibitable outward current whose amplitude was proportional to extracellular Na+ (Na+o) concentration. At all Na+o concentrations tested (3–148 mM), exchange current was maximal at negative membrane potentials (VM), but decreased as VM became more positive. Analyzing this current at each VM with a Hill equation showed that Na+–Na+ exchange had a high-affinity, low-capacity component with an apparent Na+o affinity at 0 mV (K 00.5) of 13.4 ± 0.6 mM and a low-affinity, high-capacity component with a K 00.5 of 120 ± 13 mM (n = 17). Both high- and low-affinity exchange components were VM dependent, dissipating 30 ± 3% and 82 ± 6% (n = 17) of the membrane dielectric, respectively. The low-affinity, but not the high-affinity exchange component was inhibited with 2 mM free ADP in the patch electrode solution. These results suggest that the high-affinity component of electrogenic Na+–Na+ exchange could be explained by Na+o acting as a low-affinity K + congener; however, the low-affinity component of electrogenic exchange appeared to be due to forward enzyme cycling activated by Na+o binding at a Na+-specific site deep in the membrane dielectric. A pseudo six-state model for the Na,K -ATPase was developed to simulate these data and the results of the accompanying paper (Peluffo, R.D., J.M. Argüello, and J.R. Berlin. 2000. J. Gen. Physiol. 116:47–59). This model showed that alterations in the kinetics of extracellular ion-dependent reactions alone could explain the effects of Glu779Ala substitution on the Na,K -ATPase.

Completing the uric acid degradation pathway through phylogenetic comparison of whole genomes

2006 ◽  
Vol 2 (3) ◽  
pp. 144-148 ◽  
Author(s):  
Ileana Ramazzina ◽  
Claudia Folli ◽  
Andrea Secchi ◽  
Rodolfo Berni ◽  
Riccardo Percudani
Keyword(s):  
Uric Acid ◽  
Degradation Pathway ◽  
Acid Degradation ◽  
Phylogenetic Comparison ◽  
Whole Genomes
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