scholarly journals Crystal structure and biochemical properties of putrescine carbamoyltransferase from Enterococcus faecalis : Assembly, active site, and allosteric regulation

2012 ◽  
Vol 80 (5) ◽  
pp. 1436-1447 ◽  
Author(s):  
Dashuang Shi ◽  
Xiaolin Yu ◽  
Gengxiang Zhao ◽  
Jeremy Ho ◽  
Shennon Lu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Enterococcus Faecalis ◽  
Active Site ◽  
Allosteric Regulation ◽  
Biochemical Properties

scholarly journals Structural and Functional study of Mevalonate Diphosphate Decarboxylase

2014 ◽  
Vol 70 (a1) ◽  
pp. C1636-C1636
Author(s):  
Chun-Liang Chen ◽  
Cynthia Stauffacher
Keyword(s):  
Crystal Structure ◽  
Enterococcus Faecalis ◽  
Conformational Changes ◽  
Active Site ◽  
Bacterial Infections ◽  
Mevalonate Pathway ◽  
Functional Study ◽  
Titration Calorimetry ◽  
Mevalonate Diphosphate Decarboxylase ◽  
Near Future

Mevalonate diphosphate decarboxylases (MDD) (EC 4.1.1.33) catalyze the Mg2+-dependent decarboxylation of mevalonate 5-diphosphate (MVAPP) by hydrolyzing adenosine triphosphate (ATP) and producing isopentenyl diphosphate (IPP) in the final step of mevalonate pathway. This enzyme is essential in Enterococcus faecalis and other Gram (+) bacteria; therefore, MDD protein is an ideal drug target for the treatment of bacterial infections. We have studied the enzyme kinetics and structures of MDD from Enterococcus faecalis (MDDEF) which causes clinical enterorococcal infections. In the crystal structure of the MDDEF bound with ATP, the catalytically unfavored orientation of the γ-phosphate of ATP implies that conformational changes of MDDEF might occur in order to accommodate the binding of ATP when the MVAPP binds to the active site in advance. A 10-fold decrease of the dissociation constrant (Kd) value of ATPγS has been observed using isothermal titration calorimetry (ITC) when MDDEF is pre-bound with MVAPP. The increase of binding affinity of ATPγS suggests that cooperative binding of ATP to MDDEF can be achieved by the prerequisite binding of MVAPP. Indeed, the crystal structure of MDDEF soaked with the MVAPP shows that one flexible loop that eventually should bind ATP becomes non-flexible and bends toward the active site of MDDEF. Thus, we hypothesize that the binding of the MVAPP to the active site triggers conformational changes of MDDEF which induces the binding of the other substrate, ATP, in its catalytically favored position. Further experiments will be performed for investigating a substrate-binding mechanism for MDDEF and these will serve as platforms for specific drug development in the near future.

Crystal structure and pH-dependent allosteric regulation of human β-ureidopropionase, an enzyme involved in anticancer drug metabolism*

2018 ◽  
Vol 475 (14) ◽  
pp. 2395-2416 ◽  
Author(s):  
Dirk Maurer ◽  
Bernhard Lohkamp ◽  
Michael Krumpel ◽  
Mikael Widersten ◽  
Doreen Dobritzsch
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Allosteric Regulation ◽  
Fruit Fly ◽  
Product Inhibition ◽  
Enzyme Activation ◽  
Site Directed Mutagenesis ◽  
Regulation Mechanism ◽  
Dependent Manner ◽  
Ph Dependent

β-Ureidopropionase (βUP) catalyzes the third step of the reductive pyrimidine catabolic pathway responsible for breakdown of uracil-, thymine- and pyrimidine-based antimetabolites such as 5-fluorouracil. Nitrilase-like βUPs use a tetrad of conserved residues (Cys233, Lys196, Glu119 and Glu207) for catalysis and occur in a variety of oligomeric states. Positive co-operativity toward the substrate N-carbamoyl-β-alanine and an oligomerization-dependent mechanism of substrate activation and product inhibition have been reported for the enzymes from some species but not others. Here, the activity of recombinant human βUP is shown to be similarly regulated by substrate and product, but in a pH-dependent manner. Existing as a homodimer at pH 9, the enzyme increasingly associates to form octamers and larger oligomers with decreasing pH. Only at physiological pH is the enzyme responsive to effector binding, with N-carbamoyl-β-alanine causing association to more active higher molecular mass species, and β-alanine dissociation to inactive dimers. The parallel between the pH and ligand-induced effects suggests that protonation state changes play a crucial role in the allosteric regulation mechanism. Disruption of dimer–dimer interfaces by site-directed mutagenesis generated dimeric, inactive enzyme variants. The crystal structure of the T299C variant refined to 2.08 Å resolution revealed high structural conservation between human and fruit fly βUP, and supports the hypothesis that enzyme activation by oligomer assembly involves ordering of loop regions forming the entrance to the active site at the dimer–dimer interface, effectively positioning the catalytically important Glu207 in the active site.

scholarly journals Crystal Structure, Exogenous Ligand Binding, and Redox Properties of an Engineered Diiron Active Site in a Bacterial Hemerythrin

2013 ◽  
Vol 52 (22) ◽  
pp. 13014-13020 ◽  
Author(s):  
Yasunori Okamoto ◽  
Akira Onoda ◽  
Hiroshi Sugimoto ◽  
Yu Takano ◽  
Shun Hirota ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Ligand Binding ◽  
Active Site ◽  
Redox Properties ◽  
Exogenous Ligand

scholarly journals Kinetic studies and homology modeling of a dual-substrate linalool/nerolidol synthase from Plectranthus amboinicus

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nur Suhanawati Ashaari ◽  
Mohd Hairul Ab. Rahim ◽  
Suriana Sabri ◽  
Kok Song Lai ◽  
Adelene Ai-Lian Song ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Kinetic Studies ◽  
Homology Model ◽  
Biochemical Properties ◽  
Terpene Synthase ◽  
Monoterpene Synthase ◽  
Terminal Domain ◽  
Catalytic Active Site ◽  
Plectranthus Amboinicus

AbstractLinalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C10) and farnesyl pyrophosphate (C15) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis–Menten constant (Km) and catalytic efficiency (kcat/Km) values of 16.72 ± 1.32 µM and 9.57 × 10–3 µM−1 s−1, respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx8W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase.

scholarly journals Crystal Structure of the Dithiol Oxidase DsbA Enzyme fromProteus MirabilisBound Non-covalently to an Active Site Peptide Ligand

2014 ◽  
Vol 289 (28) ◽  
pp. 19810-19822 ◽  
Author(s):  
Fabian Kurth ◽  
Wilko Duprez ◽  
Lakshmanane Premkumar ◽  
Mark A. Schembri ◽  
David P. Fairlie ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Peptide Ligand

WHICH IS THE PROTON-SHUTTLE IN ISOXANTHOPTERIN DEAMINASE? QM/MM MD UNDERSTANDING

2013 ◽  
Vol 12 (08) ◽  
pp. 1341002 ◽  
Author(s):  
XIN ZHANG ◽  
MING LEI
Keyword(s):  
Crystal Structure ◽  
Molecular Dynamics ◽  
Hydrogen Bonds ◽  
Proton Transfer ◽  
Glutamic Acid ◽  
Active Site ◽  
Nucleophilic Attack ◽  
Zinc Ions ◽  
Initial Model ◽  
Dynamics Simulations

The deamination process of isoxanthopterin catalyzed by isoxanthopterin deaminase was determined using the combined QM(PM3)/MM molecular dynamics simulations. In this paper, the updated PM3 parameters were employed for zinc ions and the initial model was built up based on the crystal structure. Proton transfer and following steps have been investigated in two paths: Asp336 and His285 serve as the proton shuttle, respectively. Our simulations showed that His285 is more effective than Aap336 in proton transfer for deamination of isoxanthopterin. As hydrogen bonds between the substrate and surrounding residues play a key role in nucleophilic attack, we suggested mutating Thr195 to glutamic acid, which could enhance the hydrogen bonds and help isoxanthopterin get close to the active site. The simulations which change the substrate to pterin 6-carboxylate also performed for comparison. Our results provide reference for understanding of the mechanism of deaminase and for enhancing the deamination rate of isoxanthopterin deaminase.

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