A shared mechanistic pathway for pyridoxal phosphate–dependent arginine oxidases

2021 ◽  
Vol 118 (40) ◽  
pp. e2012591118
Author(s):  
Elesha R. Hoffarth ◽  
Kersti Caddell Haatveit ◽  
Eugene Kuatsjah ◽  
Gregory A. MacNeil ◽  
Simran Saroya ◽  
...  
Keyword(s):  
Active Site ◽  
Evolutionary History ◽  
Pyridoxal Phosphate ◽  
Computational Studies ◽  
Single Electron Transfer ◽  
Oxidation Reactions ◽  
Unified Framework ◽  
Precise Positioning ◽  
X Ray ◽  
Mechanistic Pathway

The mechanism by which molecular oxygen is activated by the organic cofactor pyridoxal phosphate (PLP) for oxidation reactions remains poorly understood. Recent work has identified arginine oxidases that catalyze desaturation or hydroxylation reactions. Here, we investigate a desaturase from the Pseudoalteromonas luteoviolacea indolmycin pathway. Our work, combining X-ray crystallographic, biochemical, spectroscopic, and computational studies, supports a shared mechanism with arginine hydroxylases, involving two rounds of single-electron transfer to oxygen and superoxide rebound at the 4′ carbon of the PLP cofactor. The precise positioning of a water molecule in the active site is proposed to control the final reaction outcome. This proposed mechanism provides a unified framework to understand how oxygen can be activated by PLP-dependent enzymes for oxidation of arginine and elucidates a shared mechanistic pathway and intertwined evolutionary history for arginine desaturases and hydroxylases.

scholarly journals Exploring the Role of Phenylalanine Residues in Modulating the Flexibility and Topography of the Active Site in the Peroxygenase Variant PaDa-I

2020 ◽  
Vol 21 (16) ◽  
pp. 5734
Author(s):  
Joaquin Ramirez-Ramirez ◽  
Javier Martin-Diaz ◽  
Nina Pastor ◽  
Miguel Alcalde ◽  
Marcela Ayala
Keyword(s):  
Hydrogen Peroxide ◽  
Cytochrome P450 ◽  
Active Site ◽  
Bulk Solution ◽  
Computational Studies ◽  
Organic Substrates ◽  
Oxidation Reactions ◽  
Heme Group ◽  
Wide Range

Unspecific peroxygenases (UPOs) are fungal heme-thiolate enzymes able to catalyze a wide range of oxidation reactions, such as peroxidase-like, catalase-like, haloperoxidase-like, and, most interestingly, cytochrome P450-like. One of the most outstanding properties of these enzymes is the ability to catalyze the oxidation a wide range of organic substrates (both aromatic and aliphatic) through cytochrome P450-like reactions (the so-called peroxygenase activity), which involves the insertion of an oxygen atom from hydrogen peroxide. To catalyze this reaction, the substrate must access a channel connecting the bulk solution to the heme group. The composition, shape, and flexibility of this channel surely modulate the catalytic ability of the enzymes in this family. In order to gain an understanding of the role of the residues comprising the channel, mutants derived from PaDa-I, a laboratory-evolved UPO variant from Agrocybe aegerita, were obtained. The two phenylalanine residues at the surface of the channel, which regulate the traffic towards the heme active site, were mutated by less bulky residues (alanine and leucine). The mutants were experimentally characterized, and computational studies (i.e., molecular dynamics (MD)) were performed. The results suggest that these residues are necessary to reduce the flexibility of the region and maintain the topography of the channel.

A structural and mechanistic comparison of pyridoxal 5'-phosphate dependent decarboxylase and transminase enzymes

1991 ◽  
Vol 332 (1263) ◽  
pp. 131-139 ◽  
Keyword(s):  
Active Site ◽  
Pyridoxal Phosphate ◽  
Isotope Effects ◽  
Serine Hydroxymethyltransferase ◽  
Side Chain ◽  
X Ray ◽  
Kinetic Isotope ◽  
Lysine Residues ◽  
Nitrogen Nucleophiles ◽  
Active Site Histidine

Stereochemical studies of three pyridoxal phosphate dependent decarboxylases and serine hydroxymethyltransferase have allowed the dispositions of conjugate acids that operate at the C α and C-4'positions of intermediate quinoids to be determined. Kinetic work with the decarboxylase group has determined that two different acids are involved, a monoprotic acid and a polyprotic acid. The use of solvent kinetic isotope effects allowed the resolution of chemical steps in the reaction coordinate profile for decarboxylation and abortive transamination and pH-sensitivities gave the molecular p K a of the monoprotic base. Thus the ε-ammonium group of the internal aldimine-forming lysine residue operates at C-4'- si -face of the coenzyme and the imidazolium side chain of an active site histidine residue protonates at C α from the 4'- si -face. Histidine serves two other functions, as a base in generating nitrogen nucleophiles during both transaldim ination processes and as a binding group for the α-carboxyl group of substrates. The latter role for histidine was determined by comparison of the sequences for decarboxylase active site tetrapeptides (e.g. — S— X— H — K — ) with that for aspartate aminotransferase (e.g. — S— X — A— K— ) where it was known, from X-ray studies, that the serine and lysine residues interact with the coenzyme. By using the Dunathan Postulate, the conformation of the external aldimine was modified, and without changing the tetrapeptide conformation, the alanine residue was altered to a histidine. This model for the active site of a pyridoxal dependent decarboxylase was consistent with all available stereochemical and mechanistic data. A similar model for serine hydroxymethyltransferase suggested that previous reports of stereochemical infidelity with decarboxylation substrates were incorrect. A series of careful experiments confirmed this. Hence, no actual examples of non-stereospecific α-amino acid decarboxylation by pyridoxal enzymes exist.

scholarly journals Enzymatic control of dioxygen binding and functionalization of the flavin cofactor

2018 ◽  
Vol 115 (19) ◽  
pp. 4909-4914 ◽  
Author(s):  
Raspudin Saleem-Batcha ◽  
Frederick Stull ◽  
Jacob N. Sanders ◽  
Bradley S. Moore ◽  
Bruce A. Palfey ◽  
...  
Keyword(s):  
Active Site ◽  
Rational Design ◽  
Quantum Mechanical ◽  
Organic Substrates ◽  
Quantum Mechanical Calculations ◽  
Critical Transition ◽  
Precise Positioning ◽  
X Ray ◽  
X Ray Crystallography ◽  
Fine Tune

The reactions of enzymes and cofactors with gaseous molecules such as dioxygen (O2) are challenging to study and remain among the most contentious subjects in biochemistry. To date, it is largely enigmatic how enzymes control and fine-tune their reactions with O2, as exemplified by the ubiquitous flavin-dependent enzymes that commonly facilitate redox chemistry such as the oxygenation of organic substrates. Here we employ O2-pressurized X-ray crystallography and quantum mechanical calculations to reveal how the precise positioning of O2 within a flavoenzyme’s active site enables the regiospecific formation of a covalent flavin–oxygen adduct and oxygenating species (i.e., the flavin-N5-oxide) by mimicking a critical transition state. This study unambiguously demonstrates how enzymes may control the O2 functionalization of an organic cofactor as prerequisite for oxidative catalysis. Our work thus illustrates how O2 reactivity can be harnessed in an enzymatic environment and provides crucial knowledge for future rational design of O2-reactive enzymes.

One-pot Synthesis of Pyrazolone Sulfones by Iodine-catalyzed Sulfenylation of Pyrazolones with Aryl Sulfonyl Hydrazides Followed by Oxidation in Water

2018 ◽  
Vol 15 (3) ◽  
pp. 380-387
Author(s):  
Xia Zhao ◽  
Xiaoyu Lu ◽  
Lipeng Zhang ◽  
Tianjiao Li ◽  
Kui Lu
Keyword(s):  
Double Bond ◽  
Efficient Method ◽  
Room Temperature ◽  
Sulfonyl Chloride ◽  
Antibacterial Activities ◽  
Oxidation Reactions ◽  
Reaction Conditions ◽  
One Pot ◽  
X Ray ◽  
Amide Form

Aim and Objective: Pyrazolone sulfones have been reported to exhibit herbicidal and antibacterial activities. In spite of their good bioactivities, only a few methods have been developed to prepare pyrazolone sulfones. However, the substrate scope of these methods is limited. Moreover, the direct sulfonylation of pyrazolone by aryl sulfonyl chloride failed to give pyrazolone sulfones. Thus, developing a more efficient method to synthesize pyrazolone sulfones is very important. Materials and Method: Pyrazolone, aryl sulphonyl hydrazide, iodine, p-toluenesulphonic acid and water were mixed in a sealed tube, which was heated to 100°C for 12 hours. The mixture was cooled to 0°C and m-CPBA was added in batches. The mixture was allowed to stir for 30 min at room temperature. The crude product was purified by silica gel column chromatography to afford sulfuryl pyrazolone. Results: In all cases, the sulfenylation products were formed smoothly under the optimized reaction conditions, and were then oxidized to the corresponding sulfones in good yields by 3-chloroperoxybenzoic acid (m-CPBA) in water. Single crystal X-ray analysis of pyrazolone sulfone 4aa showed that the major tautomer of pyrazolone sulfones was the amide form instead of the enol form observed for pyrazolone thioethers. Moreover, the C=N double bond isomerized to form an α,β-unsaturated C=C double bond. Conclusion: An efficient method to synthesize pyrazolone thioethers by iodine-catalyzed sulfenylation of pyrazolones with aryl sulfonyl hydrazides in water was developed. Moreover, this method was employed to synthesize pyrazolone sulfones in one-pot by subsequent sulfenylation and oxidation reactions.

scholarly journals β-Ketophosphonates with Pentalenofuran Scaffolds Linked to the Ketone Group for the Synthesis of Prostaglandin Analogs

2021 ◽  
Vol 22 (13) ◽  
pp. 6787
Author(s):  
Constantin I. Tănase ◽  
Constantin Drăghici ◽  
Miron Teodor Căproiu ◽  
Anamaria Hanganu ◽  
Gheorghe Borodi ◽  
...  
Keyword(s):  
Lithium Salt ◽  
Methyl Esters ◽  
Secondary Compounds ◽  
Molecular Structures ◽  
Keto Group ◽  
High Yield ◽  
Oxidation Reactions ◽  
Prostaglandin Analogues ◽  
X Ray ◽  
Oxidation Of Alcohols

β-Ketophosphonates with pentalenofurane fragments linked to the keto group were synthesized. The bulky pentalenofurane skeleton is expected to introduce more hindrance in the prostaglandin analogues of type III, greater than that obtained with the bicyclo[3.3.0]oct(a)ene fragments of prostaglandin analogues I and II, to slow down (retard) the inactivation of the prostaglandin analogues by oxidation of 15α-OH to the 15-keto group via the 15-PGDH pathway. Their synthesis was performed by a sequence of three high yield reactions, starting from the pentalenofurane alcohols 2, oxidation of alcohols to acids 3, esterification of acids 3 to methyl esters 4 and reaction of the esters 4 with lithium salt of dimethyl methanephosphonate at low temperature. The secondary compounds 6b and 6c were formed in small amounts in the oxidation reactions of 2b and 2c, and the NMR spectroscopy showed that their structure is that of an ester of the acid with the starting alcohol. Their molecular structures were confirmed by single crystal X-ray determination method for 6c and XRPD powder method for 6b.

scholarly journals Structural basis of the stereoselective formation of the spirooxindole ring in the biosynthesis of citrinadins

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhiwen Liu ◽  
Fanglong Zhao ◽  
Boyang Zhao ◽  
Jie Yang ◽  
Joseph Ferrara ◽  
...  
Keyword(s):  
High Resolution ◽  
Organic Synthesis ◽  
Indole Alkaloids ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Directed Mutagenesis ◽  
Computational Studies ◽  
X Ray ◽  
Evolutionary Branch ◽  
Catalytic Mechanisms

AbstractPrenylated indole alkaloids featuring spirooxindole rings possess a 3R or 3S carbon stereocenter, which determines the bioactivities of these compounds. Despite the stereoselective advantages of spirooxindole biosynthesis compared with those of organic synthesis, the biocatalytic mechanism for controlling the 3R or 3S-spirooxindole formation has been elusive. Here, we report an oxygenase/semipinacolase CtdE that specifies the 3S-spirooxindole construction in the biosynthesis of 21R-citrinadin A. High-resolution X-ray crystal structures of CtdE with the substrate and cofactor, together with site-directed mutagenesis and computational studies, illustrate the catalytic mechanisms for the possible β-face epoxidation followed by a regioselective collapse of the epoxide intermediate, which triggers semipinacol rearrangement to form the 3S-spirooxindole. Comparing CtdE with PhqK, which catalyzes the formation of the 3R-spirooxindole, we reveal an evolutionary branch of CtdE in specific 3S spirocyclization. Our study provides deeper insights into the stereoselective catalytic machinery, which is important for the biocatalysis design to synthesize spirooxindole pharmaceuticals.

Structure and activity of phosphoglycerate mutase

1981 ◽  
Vol 293 (1063) ◽  
pp. 121-130 ◽  
Keyword(s):  
Active Site ◽  
Transfer Reaction ◽  
Chemical Kinetic ◽  
Phosphoglycerate Mutase ◽  
Phosphoryl Transfer ◽  
X Ray ◽  
Histidine Residues ◽  
Ping Pong ◽  
Available Information ◽  
Structure And Activity

The structure of yeast phosphoglycerate mutase determined by X-ray crystallographic and amino acid sequence studies has been interpreted in terms of the chemical, kinetic and mechanistic observations made on this enzyme. There are two histidine residues at the active site, with imidazole groups almost parallel to each other and approximately 0.4 nm apart, positioned close to the 2 and 3 positions of the substrate. The simplest interpretation of the available information suggests that a ping-pong type mechanism operates in which at least one of these histidine residues participates in the phosphoryl transfer reaction. The flexible C-terminal region also plays an important role in the enzymic reaction.

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