Interactive regulation between aliphatic hydroxylation and aromatic hydroxylation of thaxtomin D in TxtC: a theoretical investigation

ABSTRACTTxtC is an unusual bifunctional cytochrome P450 that is able to perform sequential aliphatic and aromatic hydroxylation of the diketopiperazine substrate thaxtomin D in two remote sites to produce thaxtomin A. Though the X-ray structure of TxtC complexed with thaxtomin D revealed a binding mode for its aromatic hydroxylation, the preferential hydroxylation site is aliphatic C14. It is thus intriguing to unravel how TxtC accomplishes such two-step catalytic hydroxylation on distinct aliphatic and aromatic carbons and why the aliphatic site is preferred in the hydroxylation step. In this work, by employing molecular docking and molecular dynamics (MD) simulation, we revealed that thaxtomin D could adopt two different conformations in the TxtC active site, which were equal in energy with either the aromatic C-H or aliphatic C14-H laying towards the active Cpd I oxyferryl moiety. Further ONIOM calculations indicated that the energy barrier for the rate-limiting hydroxylation step on the aliphatic C14 site was 8.9 kcal/mol more favorable than that on the aromatic C20 site. The hydroxyl group on the monohydroxylated intermediate thaxtomin B C14 site formed hydrogen bonds with Ser280 and Thr385, which induced the L-Phe moiety to rotate around the Cβ−Cγ bond of the 4-nitrotryptophan moiety. Thus, it adopted an energy favorable conformation with aromatic C20 adjacent to the oxyferryl moiety. In addition, the hydroxyl group induced solvent water molecules to enter the active site, which propelled thaxtomin B towards the heme plane and resulted in heme distortion. Based on this geometrical layout, the rate-limiting aromatic hydroxylation energy barrier decreased to 15.4 kcal/mol, which was comparable to that of the thaxtomin D aliphatic hydroxylation process. Our calculations indicated that heme distortion lowered the energy level of the lowest Cpd I α-vacant orbital, which promoted electron transfer in the rate-limiting thaxtomin B aromatic hydroxylation step in TxtC.

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Faculty Opinions recommendation of Computational prediction of structure, substrate binding mode, mechanism, and rate for a malaria protease with a novel type of active site.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1024182.290792 ◽

2005 ◽

Author(s):

Arieh Warshel

Keyword(s):

Active Site ◽

Substrate Binding ◽

Computational Prediction ◽

Binding Mode

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Recent insights into lytic polysaccharide monooxygenases (LPMOs)

Biochemical Society Transactions ◽

10.1042/bst20170549 ◽

2018 ◽

Vol 46 (6) ◽

pp. 1431-1447 ◽

Cited By ~ 39

Author(s):

Tobias Tandrup ◽

Kristian E. H. Frandsen ◽

Katja S. Johansen ◽

Jean-Guy Berrin ◽

Leila Lo Leggio

Keyword(s):

Active Site ◽

Room Temperature ◽

Experimental Studies ◽

Binding Mode ◽

Structural Features ◽

Oxygen Species ◽

Animal Kingdom ◽

X Ray ◽

Lytic Polysaccharide Monooxygenases ◽

Polysaccharide Monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are copper enzymes discovered within the last 10 years. By degrading recalcitrant substrates oxidatively, these enzymes are major contributors to the recycling of carbon in nature and are being used in the biorefinery industry. Recently, two new families of LPMOs have been defined and structurally characterized, AA14 and AA15, sharing many of previously found structural features. However, unlike most LPMOs to date, AA14 degrades xylan in the context of complex substrates, while AA15 is particularly interesting because they expand the presence of LPMOs from the predominantly microbial to the animal kingdom. The first two neutron crystallography structures have been determined, which, together with high-resolution room temperature X-ray structures, have putatively identified oxygen species at or near the active site of LPMOs. Many recent computational and experimental studies have also investigated the mechanism of action and substrate-binding mode of LPMOs. Perhaps, the most significant recent advance is the increasing structural and biochemical evidence, suggesting that LPMOs follow different mechanistic pathways with different substrates, co-substrates and reductants, by behaving as monooxygenases or peroxygenases with molecular oxygen or hydrogen peroxide as a co-substrate, respectively.

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New hypotheses for the binding mode of 4- and 7-substituted indazoles in the active site of neuronal nitric oxide synthase

Bioorganic & Medicinal Chemistry ◽

10.1016/j.bmc.2012.06.025 ◽

2012 ◽

Vol 20 (17) ◽

pp. 5296-5304 ◽

Cited By ~ 9

Author(s):

Elodie Lohou ◽

Jana Sopkova-de Oliveira Santos ◽

Pascale Schumann-Bard ◽

Michel Boulouard ◽

Silvia Stiebing ◽

...

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Active Site ◽

Neuronal Nitric Oxide Synthase ◽

Binding Mode ◽

Oxide Synthase

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Substrate specificity of sheep liver sorbitol dehydrogenase

Biochemical Journal ◽

10.1042/bj3300479 ◽

1998 ◽

Vol 330 (1) ◽

pp. 479-487 ◽

Cited By ~ 28

Author(s):

I. Rune LINDSTAD ◽

Peter KÖLL ◽

John S. McKINLEY-McKEE

Keyword(s):

Substrate Specificity ◽

Ternary Complex ◽

Kinetic Mechanism ◽

Sorbitol Dehydrogenase ◽

Hydroxyl Group ◽

Enzyme Active Site ◽

Sheep Liver ◽

Rate Limiting Step ◽

Steady State Kinetics ◽

Rate Limiting

The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo > d-ribo > L-xylo > d-lyxo ≈ l-arabino > D-arabino > l-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH >-CH2NH2 >-CH2OCH3 ≈-CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of stereospecificity at C2 in some polyols.

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An unexpected binding mode for a Pol II CTD peptide phosphorylated at Ser7 in the active site of the CTD phosphatase Ssu72

Genes & Development ◽

10.1101/gad.198853.112 ◽

2012 ◽

Vol 26 (20) ◽

pp. 2265-2270 ◽

Cited By ~ 30

Author(s):

K. Xiang ◽

J. L. Manley ◽

L. Tong

Keyword(s):

Active Site ◽

Binding Mode ◽

Pol Ii ◽

Ctd Phosphatase ◽

Phosphatase Ssu72

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Molecular Modeling and Active Site Binding Mode Characterization of Aspartate β-Semialdehyde Dehydrogenase Family

Molecular Informatics ◽

10.1002/minf.201200128 ◽

2013 ◽

Vol 32 (4) ◽

pp. 377-383 ◽

Cited By ~ 6

Author(s):

Rajender Kumar ◽

Prabha Garg

Keyword(s):

Molecular Modeling ◽

Active Site ◽

Binding Mode ◽

Semialdehyde Dehydrogenase ◽

Site Binding

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Structural Basis for the Inhibition of RNase H Activity of HIV-1 Reverse Transcriptase by RNase H Active Site-Directed Inhibitors

Journal of Virology ◽

10.1128/jvi.00353-10 ◽

2010 ◽

Vol 84 (15) ◽

pp. 7625-7633 ◽

Cited By ~ 70

Author(s):

Hua-Poo Su ◽

Youwei Yan ◽

G. Sridhar Prasad ◽

Robert F. Smith ◽

Christopher L. Daniels ◽

...

Keyword(s):

Active Site ◽

Thermal Denaturation ◽

Binding Mode ◽

Rnase H ◽

New Drugs ◽

Structural Basis ◽

Independent Manner ◽

Approved Drugs ◽

Hiv 1 ◽

Rapid Emergence

ABSTRACT HIV/AIDS continues to be a menace to public health. Several drugs currently on the market have successfully improved the ability to manage the viral burden in infected patients. However, new drugs are needed to combat the rapid emergence of mutated forms of the virus that are resistant to existing therapies. Currently, approved drugs target three of the four major enzyme activities encoded by the virus that are critical to the HIV life cycle. Although a number of inhibitors of HIV RNase H activity have been reported, few inhibit by directly engaging the RNase H active site. Here, we describe structures of naphthyridinone-containing inhibitors bound to the RNase H active site. This class of compounds binds to the active site via two metal ions that are coordinated by catalytic site residues, D443, E478, D498, and D549. The directionality of the naphthyridinone pharmacophore is restricted by the ordering of D549 and H539 in the RNase H domain. In addition, one of the naphthyridinone-based compounds was found to bind at a second site close to the polymerase active site and non-nucleoside/nucleotide inhibitor sites in a metal-independent manner. Further characterization, using fluorescence-based thermal denaturation and a crystal structure of the isolated RNase H domain reveals that this compound can also bind the RNase H site and retains the metal-dependent binding mode of this class of molecules. These structures provide a means for structurally guided design of novel RNase H inhibitors.

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Beyond the Active Site: Mechanistic Investigations of the Role of the Secondary Coordination Sphere and Beyond on Multi-Electron Electrocatalytic Reactions and Their Relations Between Kinetics and Thermodynamics

10.26434/chemrxiv.12790268 ◽

2020 ◽

Author(s):

Vincent Wang

Keyword(s):

Thermodynamic Parameters ◽

Coordination Sphere ◽

Active Site ◽

Rate Constants ◽

Reduction Reaction ◽

Rate Limiting Step ◽

Limiting Step ◽

Secondary Coordination Sphere ◽

Catalytic Rate ◽

Rate Limiting

The development of an electrocatalyst with a rapid turnover frequency, low overpotential and long-term stability is highly desired for fuel-forming reactions, such as water splitting and CO2 reduction. The findings of the scaling relationships between the catalytic rate and thermodynamic parameters over a wide range of electrocatalysts in homogeneous and heterogeneous systems provide useful guidelines and predictions for designing better catalysts for those redox reactions. However, such relationships also suggest that a catalyst with a high catalytic rate is typically associated with a high overpotential for a given reaction. Inspired by enzymes, the introduction of additional interactions through the secondary coordination sphere beyond the active site, such as hydrogen-bonding or electrostatic interactions, have been shown to offer a promising avenue to disrupt these unfavorable relationships. Herein, we further investigate the influence of these cooperative interactions on the faster chemical steps, in addition to the rate-limiting step widely examined before, for molecular electrocatalysts with the structural and electronic modifications designed to facilitate the dioxygen reduction reaction, CO2 reduction reaction and hydrogen evolving reaction. Based on the electrocatalytic kinetic analysis, the rate constants for faster chemical steps and their correlation with the corresponding thermodynamic parameters are evaluated. The results suggest that the effects of the secondary coordination sphere and beyond on these fuel-forming reactions are not necessarily beneficial for promoting all chemical steps and no apparent relation between rate constants and thermodynamic parameters are found in some cases studied here, which may implicate the design of electrocatalysts in the future. Finally, these analyses demonstrate that the characteristic features for voltammograms and foot-of-the-wave-analysis plots are associated with the specific kinetic phenomenon among these multi-electron electrocatalytic reactions, which provides a useful framework to probe the insights of chemical and electronic modifications on the catalytic steps quantitatively (i.e. kinetic rate constants) and to optimize some of critical steps beyond the rate-limiting step.

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Structure of the endonuclease IV homologue fromThermotoga maritimain the presence of active-site divalent metal ions

Acta Crystallographica Section F Structural Biology and Crystallization Communications ◽

10.1107/s1744309110028575 ◽

2010 ◽

Vol 66 (9) ◽

pp. 1003-1012 ◽

Cited By ~ 5

Author(s):

Stephen J. Tomanicek ◽

Ronny C. Hughes ◽

Joseph D. Ng ◽

Leighton Coates

Keyword(s):

Metal Ions ◽

Active Site ◽

Excision Repair ◽

Divalent Metal ◽

Hydroxyl Group ◽

Divalent Metal Ions ◽

Ap Endonuclease ◽

Phosphodiester Bond ◽

Dna Base ◽

Ap Site

The most frequent lesion in DNA is at apurinic/apyrimidinic (AP) sites resulting from DNA-base losses. These AP-site lesions can stall DNA replication and lead to genome instability if left unrepaired. The AP endonucleases are an important class of enzymes that are involved in the repair of AP-site intermediates during damage-general DNA base-excision repair pathways. These enzymes hydrolytically cleave the 5′-phosphodiester bond at an AP site to generate a free 3′-hydroxyl group and a 5′-terminal sugar phosphate using their AP nuclease activity. Specifically,Thermotoga maritimaendonuclease IV is a member of the second conserved AP endonuclease family that includesEscherichia coliendonuclease IV, which is the archetype of the AP endonuclease superfamily. In order to more fully characterize the AP endonuclease family of enzymes, two X-ray crystal structures of theT. maritimaendonuclease IV homologue were determined in the presence of divalent metal ions bound in the active-site region. These structures of theT. maritimaendonuclease IV homologue further revealed the use of the TIM-barrel fold and the trinuclear metal binding site as important highly conserved structural elements that are involved in DNA-binding and AP-site repair processes in the AP endonuclease superfamily.

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