scholarly journals A Growth-Based, High-Throughput Selection Platform Enables Remodeling of 4-Hydroxybenzoate Hydroxylase Active Site

ACS Catalysis ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 6969-6974 ◽  
Author(s):  
Sarah Maxel ◽  
Derek Aspacio ◽  
Edward King ◽  
Linyue Zhang ◽  
Ana Paula Acosta ◽  
...  
Keyword(s):  
High Throughput ◽  
Active Site ◽  
Hydroxybenzoate Hydroxylase

scholarly journals A growth-based, high-throughput selection platform enables remodeling of 4-hydroxybenzoate hydroxylase active site

2020 ◽  
Author(s):  
Sarah Maxel ◽  
Derek Aspacio ◽  
Edward King ◽  
Linyue Zhang ◽  
Ana Paula Acosta ◽  
...  
Keyword(s):  
High Throughput ◽  
Active Site ◽  
Catalytic Efficiency ◽  
Redox Balance ◽  
Dihydroxybenzoic Acid ◽  
Aerobic Growth ◽  
Acid Biosynthesis ◽  
Bond Network ◽  
Hydroxybenzoate Hydroxylase ◽  
Universal Application

ABSTRACTWe report an aerobic, growth-based selection platform founded on NADP(H) redox balance restoration in Escherichia coli, and demonstrate its application in high-throughput evolution of oxygenase. A single round of selection enabled Pseudomonas aeruginoasa 4-hydroxybenzoate hydroxylase (PobA) to accept 3,4-dihydroxybenzoic acid efficiently, an essential step toward gallic acid biosynthesis. The best variant DA015 exhibited more than 5-fold higher catalytic efficiency compared to previously engineered enzymes. Structural modeling suggests precise re-organization of active site hydrogen bond network, which is difficult to obtain without deep navigation of combinatorial sequence space. We envision universal application of this selection platform in engineering NADPH-dependent oxidoreductases.

scholarly journals High-throughput quantum-mechanics/molecular-mechanics (ONIOM) macromolecular crystallographic refinement withPHENIX/DivCon: the impact of mixed Hamiltonian methods on ligand and protein structure

2018 ◽  
Vol 74 (11) ◽  
pp. 1063-1077 ◽  
Author(s):  
Oleg Borbulevych ◽  
Roger I. Martin ◽  
Lance M. Westerhoff
Keyword(s):  
Quantum Mechanics ◽  
Molecular Mechanics ◽  
High Throughput ◽  
Active Site ◽  
Binding Modes ◽  
Acta Cryst ◽  
Structure Based Drug Design ◽  
Energy Functionals ◽  
Conventional Methods ◽  
The Impact

Conventional macromolecular crystallographic refinement relies on often dubious stereochemical restraints, the preparation of which often requires human validation for unusual species, and on rudimentary energy functionals that are devoid of nonbonding effects owing to electrostatics, polarization, charge transfer or even hydrogen bonding. While this approach has served the crystallographic community for decades, as structure-based drug design/discovery (SBDD) has grown in prominence it has become clear that these conventional methods are less rigorous than they need to be in order to produce properly predictive protein–ligand models, and that the human intervention that is required to successfully treat ligands and other unusual chemistries found in SBDD often precludes high-throughput, automated refinement. Recently, plugins to thePython-based Hierarchical ENvironment for Integrated Xtallography(PHENIX) crystallographic platform have been developed to augment conventional methods with thein situuse of quantum mechanics (QM) applied to ligand(s) along with the surrounding active site(s) at each step of refinement [Borbulevychet al.(2014),Acta CrystD70, 1233–1247]. This method (Region-QM) significantly increases the accuracy of the X-ray refinement process, and this approach is now used, coupled with experimental density, to accurately determine protonation states, binding modes, ring-flip states, water positions and so on. In the present work, this approach is expanded to include a more rigorous treatment of the entire structure, including the ligand(s), the associated active site(s) and the entire protein, using a fully automated, mixed quantum-mechanics/molecular-mechanics (QM/MM) Hamiltonian recently implemented in theDivConpackage. This approach was validated through the automatic treatment of a population of 80 protein–ligand structures chosen from the Astex Diverse Set. Across the entire population, this method results in an average 3.5-fold reduction in ligand strain and a 4.5-fold improvement inMolProbityclashscore, as well as improvements in Ramachandran and rotamer outlier analyses. Overall, these results demonstrate that the use of a structure-wide QM/MM Hamiltonian exhibits improvements in the local structural chemistry of the ligand similar to Region-QM refinement but with significant improvements in the overall structure beyond the active site.

scholarly journals Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

Science ◽  
2021 ◽  
Vol 373 (6553) ◽  
pp. eabf8761
Author(s):  
C. J. Markin ◽  
D. A. Mokhtari ◽  
F. Sunden ◽  
M. J. Appel ◽  
E. Akiva ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Enzyme Kinetics ◽  
High Throughput ◽  
Active Site ◽  
Molecular Mechanisms ◽  
Extensive Investigation ◽  
Purification And Characterization ◽  
Multiple Substrates ◽  
Catalytic Effects

Systematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We present HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), a microfluidic platform for high-throughput expression, purification, and characterization of more than 1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA (phosphate-irrepressible alkaline phosphatase of Flavobacterium), we performed more than 670,000 reactions and determined more than 5000 kinetic and physical constants for multiple substrates and inhibitors. We uncovered extensive kinetic partitioning to a misfolded state and isolated catalytic effects, revealing spatially contiguous regions of residues linked to particular aspects of function. Regions included active-site proximal residues but extended to the enzyme surface, providing a map of underlying architecture not possible to derive from existing approaches. HT-MEK has applications that range from understanding molecular mechanisms to medicine, engineering, and design.

The coenzyme analog adenosine 5-diphosphoribose displaces FAD in the active site of p-hydroxybenzoate hydroxylase. An x-ray crystallographic investigation

Biochemistry ◽  
1989 ◽  
Vol 28 (18) ◽  
pp. 7199-7205 ◽  
Author(s):  
Jan M. Van der Laan ◽  
Herman A. Schreuder ◽  
Myra B. A. Swarte ◽  
Rik K. Wierenga ◽  
Kor H. Kalk ◽  
...  
Keyword(s):  
Active Site ◽  
Crystallographic Investigation ◽  
X Ray ◽  
Hydroxybenzoate Hydroxylase

pH-Dependent Structural Changes in the Active Site ofp-Hydroxybenzoate Hydroxylase Point to the Importance of Proton and Water Movements during Catalysis†,‡

Biochemistry ◽  
1996 ◽  
Vol 35 (2) ◽  
pp. 567-578 ◽  
Author(s):  
Domenico L. Gatti ◽  
Barrie Entsch ◽  
David P. Ballou ◽  
Martha L. Ludwig
Keyword(s):  
Active Site ◽  
Structural Changes ◽  
Ph Dependent ◽  
Hydroxybenzoate Hydroxylase

scholarly journals Influence of Fluorescent Probe Size and Cytochrome b5 on Drug-Drug Interactions in CYP2C9

2006 ◽  
Vol 11 (3) ◽  
pp. 303-309 ◽  
Author(s):  
Matthew A. Hummel ◽  
Timothy S. Tracy ◽  
J. Matthew Hutzler ◽  
Jan L. Wahlstrom ◽  
Yihong Zhou ◽  
...  
Keyword(s):  
Drug Interaction ◽  
Drug Interactions ◽  
Fluorescent Probe ◽  
High Throughput ◽  
Active Site ◽  
Cytochrome B5 ◽  
Limited Impact ◽  
Probe Size ◽  
Multiple Substrate ◽  
High Throughput Screens

7-Methoxy-4-trifluoromethylcoumarin (MFC) has been used extensively in high-throughput screens for the identification of potential CYP2C9 interactions. More recently, additional probes from Invitrogen have been used. Vivid 2C9Green is the largest of the probes and has had limited prior characterization. The newseries of probes differ significantly from MFC andwere examined for their ability to identify interactions with 19 CYP2C9 substrates/inhibitors. The inhibition profiles depend largely on the physical differences between the fluorescent probe substrates. Cytochrome b5 (cyt b5) was also investigated for the ability to alter the inhibition profile of a given compound. The stoichiometric addition of cyt b5 caused an increase in Vmaxof MFC and Vivid 2C9 Green 4.4 and 1.7 times, respectively. Furthermore, cyt b5 imposes a steric component to the active site as the inhibition profiles were significantly affected in incubations with MFC. The addition of cyt b5 had limited impact on the inhibition profiles generated with Vivid 2C9Green. The Km of Vivid 2C9 Green increased from 1.2 ± 0.2 µ Mto4.8 ± 0.3 µ Mas a result of cyt b5 addition. These results illustrate that multiple substrate probes may be necessary for screening drug-drug interaction in CYP2C9 and that cyt b5 effects can impart steric restraints on the CYP2C9 active site.

scholarly journals Synthesis of triazole-containing ciprofloxacin conjugate and its in silico test as a cytochrome P450 ligand

2021 ◽  
pp. 21-27
Author(s):  
Ярослав Вячеславович Фалетров ◽  
Лилия Игоревна Глинская ◽  
Матвей Сергеевич Хорецкий ◽  
Ян Владимирович Панада ◽  
Нина Степановна Фролова ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Virtual Screening ◽  
High Throughput ◽  
Active Site ◽  
Essential Role ◽  
Cytochromes P450 ◽  
Heme Iron ◽  
Cellular Functions ◽  
Docking Calculations

Cytochromes P450 are hem-containing monooxygenases which catalyse biosynthesis of many compounds playing an essential role in cellular functions as well as degradation of drugs and xenobiotics. Some P450s (e. g., human CYP19 and CYP17, fungal CYP51) are valid target proteins for some drugs. The others P450s are also interesting for pharmacology-related researches. Aiming to design new fluorescent inhibitor of P450s we have synthesised the azole-bearing conjugate of ciprofloxacin (CPF-bab-Z1). To estimate potential of the compound as a ligand for CYPs we performed high-throughput virtual screening (multiple docking calculations) for CPF-bab-Z1 and multiple known 3D structures of P450s. The best affinity for CPF-bab-Z1 (the smallest value of energy of binding is equal -12.5 kcal/mol) were found for protein with PDB code 5esh among 28 structures of CYP51. The calculated pose of CPF-bab-Z1 in the active site of the protein is characterised by cyclopropyl (but not azole) proximity to the heme iron of the CYP51. The data obtained demonstrate perspectives for in vitro investigations of CPF-bab-Z1 with P450s.

Crystal Structure of 3-Hydroxybenzoate Hydroxylase from Comamonas testosteroni Has a Large Tunnel for Substrate and Oxygen Access to the Active Site

2006 ◽  
Vol 364 (5) ◽  
pp. 878-896 ◽  
Author(s):  
Takeshi Hiromoto ◽  
Shinsuke Fujiwara ◽  
Keiichi Hosokawa ◽  
Hiroshi Yamaguchi
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Comamonas Testosteroni ◽  
Hydroxybenzoate Hydroxylase ◽  
Large Tunnel

scholarly journals Indoline-6-Sulfonamide Inhibitors of the Bacterial Enzyme DapE

Antibiotics ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 595
Author(s):  
Cory T. Reidl ◽  
Tahirah K. Heath ◽  
Iman Darwish ◽  
Rachel M. Torrez ◽  
Maxwell Moore ◽  
...  
Keyword(s):  
Molecular Docking ◽  
High Throughput ◽  
Mechanism Of Action ◽  
Active Site ◽  
Diaminopimelic Acid ◽  
Zinc Binding ◽  
High Throughput Screen ◽  
Inhibitory Potency ◽  
Bacterial Enzyme ◽  
Site Binding

Inhibitors of the bacterial enzyme dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE; EC 3.5.1.18) hold promise as antibiotics with a new mechanism of action. Herein we describe the discovery of a new series of indoline sulfonamide DapE inhibitors from a high-throughput screen and the synthesis of a series of analogs. Inhibitory potency was measured by a ninhydrin-based DapE assay recently developed by our group. Molecular docking experiments suggest active site binding with the sulfonamide acting as a zinc-binding group (ZBG).

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