Removal of the Side Chain at the Active-Site Serine by a Glycine Substitution Increases the Stability of a Wide Range of Serine β-Lactamases by Relieving Steric Strain

3-D Structure of Serum Paraoxonase 1 Sheds Light on Its Activity, Stability, Solubility and CrystallizabilitySerum paraoxonases (PONs) exhibit a wide range of physiologically important hydrolytic activities, including drug metabolism and detoxification of nerve gases. PON1 and PON3 reside on high-density lipoprotein (HDL) (the "good cholesterol"), and are involved in the alleviation of atherosclerosis. Members of the PON family have been identified not only in mammals and other vertebrates, but also in invertebrates. We earlier described the first crystal structure of a PON family member, a directly-evolved variant of PON1, at 2.2 Å resolution. PON1 is a 6-bladed beta-propeller with a unique active-site lid which is also involved in binding to HDL. The 3-D structure, taken together with directed evolution studies, permitted analysis of mutations which enhanced the stability, solubility and crystallizability of this PON1 variant. The structure permits a detailed description of PON1's active site and suggests possible mechanisms for its catalytic activity on certain substrates.

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Oxidation by 2-oxoglutarate oxygenases: non-haem iron systems in catalysis and signalling

Philosophical Transactions of The Royal Society A Mathematical Physical and Engineering Sciences ◽

10.1098/rsta.2004.1540 ◽

2005 ◽

Vol 363 (1829) ◽

pp. 807-828 ◽

Cited By ~ 48

Author(s):

K.S Hewitson ◽

N Granatino ◽

R.W.D Welford ◽

M.A McDonough ◽

C.J Schofield

Keyword(s):

Active Site ◽

Ferrous Iron ◽

Substrate Oxidation ◽

Conformational Flexibility ◽

Side Chain ◽

Oxidative Decarboxylation ◽

Structural Studies ◽

Wide Range ◽

Ferryl Species ◽

Haem Iron

The 2-oxoglutarate (2OG) and ferrous iron dependent oxygenases are a superfamily of enzymes that catalyse a wide range of reactions including hydroxylations, desaturations and oxidative ring closures. Recently, it has been discovered that they act as sensors in the hypoxic response in humans and other animals. Substrate oxidation is coupled to conversion of 2OG to succinate and carbon dioxide. Kinetic, spectroscopic and structural studies are consistent with a consensus mechanism in which ordered binding of (co)substrates enables control of reactive intermediates. Binding of the substrate to the active site triggers the enzyme for ligation of dioxygen to the metal. Oxidative decarboxylation of 2OG then generates the ferryl species thought to mediate substrate oxidation. Structural studies reveal a conserved double-stranded β-helix core responsible for binding the iron, via a 2His-1carboxylate motif and the 2OG side chain. The rigidity of this core contrasts with the conformational flexibility of surrounding regions that are involved in binding the substrate. Here we discuss the roles of 2OG oxygenases in terms of the generic structural and mechanistic features that render the 2OG oxygenases suited for their functions.

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Performance of Electronic Structure Methods for the Description of Hückel-Möbius Interconversions in Extended π-Systems

10.26434/chemrxiv.10564115 ◽

2019 ◽

Author(s):

Tatiana Woller ◽

Ambar Banerjee ◽

Nitai Sylvetsky ◽

Xavier Deraet ◽

Frank De Proft ◽

...

Keyword(s):

Electronic Structure ◽

Basis Set ◽

Dft Methods ◽

Molecular Switching ◽

Wide Range ◽

Electronic Structure Methods ◽

Explicitly Correlated ◽

Relative Errors ◽

The Stability ◽

Basis Set Expansion

Expanded porphyrins provide a versatile route to molecular switching devices due to their ability to shift between several π-conjugation topologies encoding distinct properties. Taking into account its size and huge conformational flexibility, DFT remains the workhorse for modeling such extended macrocycles. Nevertheless, the stability of Hückel and Möbius conformers depends on a complex interplay of different factors, such as hydrogen bonding, p···p stacking, steric effects, ring strain and electron delocalization. As a consequence, the selection of an exchange-correlation functional for describing the energy profile of topological switches is very difficult. For these reasons, we have examined the performance of a variety of wavefunction methods and density functionals for describing the thermochemistry and kinetics of topology interconversions across a wide range of macrocycles. Especially for hexa- and heptaphyrins, the Möbius structures have a pronouncedly stronger degree of static correlation than the Hückel and figure-eight structures, and as a result the relative energies of singly-twisted structures are a challenging test for electronic structure methods. Comparison of limited orbital space full CI calculations with CCSD(T) calculations within the same active spaces shows that post-CCSD(T) correlation contributions to relative energies are very minor. At the same time, relative energies are weakly sensitive to further basis set expansion, as proven by the minor energy differences between MP2/cc-pVDZ and explicitly correlated MP2-F12/cc-pVDZ-F12 calculations. Hence, our CCSD(T) reference values are reasonably well-converged in both 1-particle and n-particle spaces. While conventional MP2 and MP3 yield very poor results, SCS-MP2 and particularly SOS-MP2 and SCS-MP3 agree to better than 1 kcal mol-1 with the CCSD(T) relative energies. Regarding DFT methods, only M06-2X provides relative errors close to chemical accuracy with a RMSD of 1.2 kcal mol-1. While the original DSD-PBEP86 double hybrid performs fairly poorly for these extended p-systems, the errors drop down to 2 kcal mol-1 for the revised revDSD-PBEP86-NL, again showing that same-spin MP2-like correlation has a detrimental impact on performance like the SOS-MP2 results.

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Strongly Coupled Redox-Linked Conformational Switching at the Active Site of the Non-Heme Iron-Dependent Dioxygenase, TauD

10.26434/chemrxiv.9461462 ◽

2019 ◽

Author(s):

Christopher John ◽

Greg M. Swain ◽

Robert P. Hausinger ◽

Denis A. Proshlyakov

Keyword(s):

Active Site ◽

Structural Model ◽

Heme Iron ◽

Chemical Transformations ◽

Experimental Conditions ◽

Strongly Coupled ◽

Non Heme Iron ◽

Reversible Redox ◽

Wide Range ◽

Complex Structural

2-Oxoglutarate (2OG)-dependent dioxygenases catalyze C-H activation while performing a wide range of chemical transformations. In contrast to their heme analogues, non-heme iron centers afford greater structural flexibility with important implications for their diverse catalytic mechanisms. We characterize an in situ structural model of the putative transient ferric intermediate of 2OG:taurine dioxygenase (TauD) by using a combination of spectroelectrochemical and semi-empirical computational methods, demonstrating that the Fe (III/II) transition involves a substantial, fully reversible, redox-linked conformational change at the active site. This rearrangement alters the apparent redox potential of the active site between -127 mV for reduction of the ferric state and 171 mV for oxidation of the ferrous state of the 2OG-Fe-TauD complex. Structural perturbations exhibit limited sensitivity to mediator concentrations and potential pulse duration. Similar changes were observed in the Fe-TauD and taurine-2OG-Fe-TauD complexes, thus attributing the reorganization to the protein moiety rather than the cosubstrates. Redox difference infrared spectra indicate a reorganization of the protein backbone in addition to the involvement of carboxylate and histidine ligands. Quantitative modeling of the transient redox response using two alternative reaction schemes across a variety of experimental conditions strongly supports the proposal for intrinsic protein reorganization as the origin of the experimental observations.

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Insights into Potent Therapeutical Antileukemic Agent L-glutaminase Enzyme Under Solid-state Fermentation: A Review

Current Drug Metabolism ◽

10.2174/1389200221666200421122147 ◽

2020 ◽

Vol 21 (3) ◽

pp. 211-220 ◽

Cited By ~ 1

Author(s):

Chandrasai Potla Durthi ◽

Madhuri Pola ◽

Satish Babu Rajulapati ◽

Anand Kishore Kola

Keyword(s):

Solid State ◽

Solid State Fermentation ◽

Food Industry ◽

Energy Requirement ◽

Production Studies ◽

Wide Range ◽

Hybridoma Technology ◽

Bacteria And Fungi ◽

The Stability ◽

Glutaminase Activity

Aim & objective: To review the applications and production studies of reported antileukemic drug L-glutaminase under Solid-state Fermentation (SSF). Overview: An amidohydrolase that gained economic importance because of its wide range of applications in the pharmaceutical industry, as well as the food industry, is L-glutaminase. The medical applications utilized it as an anti-tumor agent as well as an antiretroviral agent. L-glutaminase is employed in the food industry as an acrylamide degradation agent, as a flavor enhancer and for the synthesis of theanine. Another application includes its use in hybridoma technology as a biosensing agent. Because of its diverse applications, scientists are now focusing on enhancing the production and optimization of L-glutaminase from various sources by both Solid-state Fermentation (SSF) and submerged fermentation studies. Of both types of fermentation processes, SSF has gained importance because of its minimal cost and energy requirement. L-glutaminase can be produced by SSF from both bacteria and fungi. Single-factor studies, as well as multi-level optimization studies, were employed to enhance L-glutaminase production. It was concluded that L-glutaminase activity achieved by SSF was 1690 U/g using wheat bran and Bengal gram husk by applying feed-forward artificial neural network and genetic algorithm. The highest L-glutaminase activity achieved under SSF was 3300 U/gds from Bacillus sp., by mixture design. Purification and kinetics studies were also reported to find the molecular weight as well as the stability of L-glutaminase. Conclusion: The current review is focused on the production of L-glutaminase by SSF from both bacteria and fungi. It was concluded from reported literature that optimization studies enhanced L-glutaminase production. Researchers have also confirmed antileukemic and anti-tumor properties of the purified L-glutaminase on various cell lines.

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Polymer-Drug Conjugates as Nanotheranostic Agents

Journal of Nanotheranostics ◽

10.3390/jnt2010005 ◽

2021 ◽

Vol 2 (1) ◽

pp. 63-81

Author(s):

Sajana Manandhar ◽

Erica Sjöholm ◽

Johan Bobacka ◽

Jessica M. Rosenholm ◽

Kuldeep K. Bansal

Keyword(s):

Drug Delivery ◽

Drug Release ◽

Treatment Options ◽

The Body ◽

Drug Conjugates ◽

Imaging Characteristics ◽

Wide Range ◽

Systemic Side Effects ◽

Theranostic Agents ◽

The Stability

Since the last decade, the polymer-drug conjugate (PDC) approach has emerged as one of the most promising drug-delivery technologies owing to several benefits like circumventing premature drug release, offering controlled and targeted drug delivery, improving the stability, safety, and kinetics of conjugated drugs, and so forth. In recent years, PDC technology has advanced with the objective to further enhance the treatment outcomes by integrating nanotechnology and multifunctional characteristics into these systems. One such development is the ability of PDCs to act as theranostic agents, permitting simultaneous diagnosis and treatment options. Theranostic nanocarriers offer the opportunity to track the distribution of PDCs within the body and help to localize the diseased site. This characteristic is of particular interest, especially among those therapeutic approaches where external stimuli are supposed to be applied for abrupt drug release at the target site for localized delivery to avoid systemic side effects (e.g., Visudyne®). Thus, with the help of this review article, we are presenting the most recent updates in the domain of PDCs as nanotheranostic agents. Different methodologies utilized to design PDCs along with imaging characteristics and their applicability in a wide range of diseases, have been summarized in this article.

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No-z Model for Magnetic Fields of Different Astrophysical Objects and Stability of the Solutions

Data ◽

10.3390/data6010004 ◽

2021 ◽

Vol 6 (1) ◽

pp. 4

Author(s):

Evgeny Mikhailov ◽

Daniela Boneva ◽

Maria Pashentseva

Keyword(s):

Magnetic Field ◽

Magnetic Fields ◽

Large Scale ◽

Point Of View ◽

Accretion Discs ◽

Wide Range ◽

Astrophysical Objects ◽

Alpha Effect ◽

The Stability ◽

The Magnetic Field

A wide range of astrophysical objects, such as the Sun, galaxies, stars, planets, accretion discs etc., have large-scale magnetic fields. Their generation is often based on the dynamo mechanism, which is connected with joint action of the alpha-effect and differential rotation. They compete with the turbulent diffusion. If the dynamo is intensive enough, the magnetic field grows, else it decays. The magnetic field evolution is described by Steenbeck—Krause—Raedler equations, which are quite difficult to be solved. So, for different objects, specific two-dimensional models are used. As for thin discs (this shape corresponds to galaxies and accretion discs), usually, no-z approximation is used. Some of the partial derivatives are changed by the algebraic expressions, and the solenoidality condition is taken into account as well. The field generation is restricted by the equipartition value and saturates if the field becomes comparable with it. From the point of view of mathematical physics, they can be characterized as stable points of the equations. The field can come to these values monotonously or have oscillations. It depends on the type of the stability of these points, whether it is a node or focus. Here, we study the stability of such points and give examples for astrophysical applications.

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Active site-directed inhibitors of cytochrome P-450scc. Structural and mechanistic implications of a side chain-substituted series of amino-steroids.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)44246-3 ◽

1983 ◽

Vol 258 (19) ◽

pp. 11446-11452 ◽

Cited By ~ 4

Author(s):

J J Sheets ◽

L E Vickery

Keyword(s):

Active Site ◽

Side Chain

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Chemical modification of serine at the active site of penicillin acylase from Kluyvera citrophila

Biochemical Journal ◽

10.1042/bj2800659 ◽

1991 ◽

Vol 280 (3) ◽

pp. 659-662 ◽

Cited By ~ 15

Author(s):

J Martín ◽

A Slade ◽

A Aitken ◽

R Arche ◽

R Virden

Keyword(s):

Active Site ◽

Tertiary Structure ◽

Thiol Group ◽

Iodoacetic Acid ◽

Penicillin Acylase ◽

Protein Product ◽

Side Chain ◽

Serine Residue ◽

Conserved Sequence ◽

Ester Substrate

The site of reaction of penicillin acylase from Kluyvera citrophila with the potent inhibitor phenylmethanesulphonyl fluoride was investigated by incubating the inactivated enzyme with thioacetic acid to convert the side chain of the putative active-site serine residue to that of cysteine. The protein product contained one thiol group, which was reactive towards 2,2′-dipyridyl disulphide and iodoacetic acid. Carboxymethylcysteine was identified as the N-terminal residue of the beta-subunit of the carboxy[3H]methylthiol-protein. No significant changes in tertiary structure were detected in the modified penicillin acylase using near-u.v. c.d. spectroscopy. However, the catalytic activity (kcat) with either an anilide or an ester substrate was decreased in the thiol-protein by a factor of more than 10(4). A comparison of sequences of apparently related acylases shows no other extensive regions of conserved sequence containing an invariant serine residue. The side chain of this residue is proposed as a candidate nucleophile in the formation of an acyl-enzyme during catalysis.

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