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Mechanism of Enzyme Catalysis: When Structure Meets Function

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Biochemistry".

Deadline for manuscript submissions: 25 October 2024 | Viewed by 4665

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Dr. Ivana Leščić Ašler

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Laboratory for Chemical and Biological Crystallography, Division of Physical Chemistry, Ruđer Bošković Institute, Bijenička cesta 54, HR-10 000 Zagreb, Croatia
Interests: metallosensor proteins from pathogenic and non-pathogenic bacteria; purine salvage pathway enzymes from Escherichia coli and Helicobacter pylori; bacterial SGNH hydrolases; mechanisms of allosteric regulation; protein-DNA binding; protein crystallography; protein mass spectrometry; enzyme kinetics and regulation; substrate specificity and promiscuity

Special Issue Information

Dear Colleagues,

Proteins are essential biomacromolecules in all organisms, as they perform diverse biochemical tasks in cells, including enzymatic catalysis. Advances in sequencing technologies and computational power provided an explosion of sequence information—currently there are almost 250 million of nucleotide sequences in GenBank database. However, only ~570 000 of protein sequences are listed in SwissProt, the manually annotated and reviewed portion of UniProtKB, and only a small percentage of these have experimentally determined function. The function of proteins, and more specifically enzymes, is intimately linked with their three-dimensional structure. The size, shape and charge of the active site, interactions between domains and/or subunits, protein dynamics, existence of cofactor and/or allosteric sites, conservation of catalytic residues, these are just some of the key structural features that reveal mechanistic details of molecular function and lead to hypothesis about how a given enzyme operates. One has to keep in mind, though, that structure alone is not enough to predict specific function of an enzyme. As enzyme catalysis can be finely tuned by e.g. minute differences in active site or allosteric site residues, substrate specificity determination requires functional studies (i.e., old-fashioned biochemistry). For an enzyme to be applicable in any branch of industry or to be considered as a drug target, its structure and function have to be investigated in detail, and by a multidisciplinary approach, collecting the knowledge from all possible sides. Following this line of thought, this special issue welcomes contributions containing both experimental and computational research aspiring to shed a light on the complex topic of the mechanism of enzyme catalysis.

Dr. Ivana Leščić Ašler
Guest Editor

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Keywords

three-dimensional structure of enzymes
enzyme stability, solubility and flexibility
structure-function relationship in enzymes
mechanism of enzyme catalysis
enzyme kinetics
substrate specificity of enzymes
regulation of enzyme activity
enzyme engineering

Published Papers (6 papers)

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Research

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20 pages, 5469 KiB

Open AccessArticle

Location Is Everything: Influence of His-Tag Fusion Site on Properties of Adenylosuccinate Synthetase from Helicobacter pylori

by Marija Zora Mišković, Marta Wojtyś, Maria Winiewska-Szajewska, Beata Wielgus-Kutrowska, Marija Matković, Darija Domazet Jurašin, Zoran Štefanić, Agnieszka Bzowska and Ivana Leščić Ašler

Int. J. Mol. Sci. 2024, 25(14), 7613; https://doi.org/10.3390/ijms25147613 - 11 Jul 2024

Viewed by 321

Abstract

The requirement for fast and dependable protein purification methods is constant, either for functional studies of natural proteins or for the production of biotechnological protein products. The original procedure has to be formulated for each individual protein, and this demanding task was significantly simplified by the introduction of affinity tags. Helicobacter pylori adenylosuccinate synthetase (AdSS) is present in solution in a dynamic equilibrium of monomers and biologically active homodimers. The addition of the His₆-tag on the C-terminus (C-His-AdSS) was proven to have a negligible effect on the characteristics of this enzyme. This paper shows that the same enzyme with the His₆-tag fused on its N-terminus (N-His-AdSS) has a high tendency to precipitate. Circular dichroism and X-ray diffraction studies do not detect any structural change that could explain this propensity. However, the dynamic light scattering, differential scanning fluorimetry, and analytical ultracentrifugation measurements indicate that the monomer of this construct is prone to aggregation, which shifts the equilibrium towards the insoluble precipitant. In agreement, enzyme kinetics measurements showed reduced enzyme activity, but preserved affinity for the substrates, in comparison with the wild-type and C-His-AdSS. The presented results reinforce the notion that testing the influence of the tag on protein properties should not be overlooked. Full article

(This article belongs to the Special Issue Mechanism of Enzyme Catalysis: When Structure Meets Function)

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14 pages, 4552 KiB

Open AccessArticle

Molecular Docking Studies of Ortho-Substituted Phenols to Tyrosinase Helps Discern If a Molecule Can Be an Enzyme Substrate

by María F. Montenegro, José A. Teruel, Pablo García-Molina, José Tudela, José Neptuno Rodríguez-López, Francisco García-Cánovas and Francisco García-Molina

Int. J. Mol. Sci. 2024, 25(13), 6891; https://doi.org/10.3390/ijms25136891 - 23 Jun 2024

Viewed by 512

Abstract

Phenolic compounds with a position ortho to the free phenolic hydroxyl group occupied can be tyrosinase substrates. However, ortho-substituted compounds are usually described as inhibitors. The mechanism of action of tyrosinase on monophenols is complex, and if they are ortho-substituted, it is more complicated. It can be shown that many of these molecules can become substrates of the enzyme in the presence of catalytic o-diphenol, MBTH, or in the presence of hydrogen peroxide. Docking studies can help discern whether a molecule can behave as a substrate or inhibitor of the enzyme. Specifically, phenols such as thymol, carvacrol, guaiacol, eugenol, isoeugenol, and ferulic acid are substrates of tyrosinase, and docking simulations to the active center of the enzyme predict this since the distance of the peroxide oxygen from the oxy-tyrosinase form to the ortho position of the phenolic hydroxyl is adequate for the electrophilic attack reaction that gives rise to hydroxylation occurring. Full article

(This article belongs to the Special Issue Mechanism of Enzyme Catalysis: When Structure Meets Function)

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16 pages, 6234 KiB

Open AccessArticle

Structural Evaluation of a Nitroreductase Engineered for Improved Activation of the 5-Nitroimidazole PET Probe SN33623

by Abigail V. Sharrock, Jeff S. Mumm, Elsie M. Williams, Narimantas Čėnas, Jeff B. Smaill, Adam V. Patterson, David F. Ackerley, Gintautas Bagdžiūnas and Vickery L. Arcus

Int. J. Mol. Sci. 2024, 25(12), 6593; https://doi.org/10.3390/ijms25126593 - 15 Jun 2024

Viewed by 512

Abstract

Bacterial nitroreductase enzymes capable of activating imaging probes and prodrugs are valuable tools for gene-directed enzyme prodrug therapies and targeted cell ablation models. We recently engineered a nitroreductase (E. coli NfsB F70A/F108Y) for the substantially enhanced reduction of the 5-nitroimidazole PET-capable probe, SN33623, which permits the theranostic imaging of vectors labeled with oxygen-insensitive bacterial nitroreductases. This mutant enzyme also shows improved activation of the DNA-alkylation prodrugs CB1954 and metronidazole. To elucidate the mechanism behind these enhancements, we resolved the crystal structure of the mutant enzyme to 1.98 Å and compared it to the wild-type enzyme. Structural analysis revealed an expanded substrate access channel and new hydrogen bonding interactions. Additionally, computational modeling of SN33623, CB1954, and metronidazole binding in the active sites of both the mutant and wild-type enzymes revealed key differences in substrate orientations and interactions, with improvements in activity being mirrored by reduced distances between the N5-H of isoalloxazine and the substrate nitro group oxygen in the mutant models. These findings deepen our understanding of nitroreductase substrate specificity and catalytic mechanisms and have potential implications for developing more effective theranostic imaging strategies in cancer treatment. Full article

(This article belongs to the Special Issue Mechanism of Enzyme Catalysis: When Structure Meets Function)

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12 pages, 2239 KiB

Open AccessArticle

Mechanistic and Structural Insights on Difluoromethyl-1,3,4-oxadiazole Inhibitors of HDAC6

by Edoardo Cellupica, Aureliano Gaiassi, Ilaria Rocchio, Grazia Rovelli, Roberta Pomarico, Giovanni Sandrone, Gianluca Caprini, Paola Cordella, Cyprian Cukier, Gianluca Fossati, Mattia Marchini, Aleksandra Bebel, Cristina Airoldi, Alessandro Palmioli, Andrea Stevenazzi, Christian Steinkühler and Barbara Vergani

Int. J. Mol. Sci. 2024, 25(11), 5885; https://doi.org/10.3390/ijms25115885 - 28 May 2024

Viewed by 606

Abstract

Histone deacetylase 6 (HDAC6) is increasingly recognized for its potential in targeted disease therapy. This study delves into the mechanistic and structural nuances of HDAC6 inhibition by difluoromethyl-1,3,4-oxadiazole (DFMO) derivatives, a class of non-hydroxamic inhibitors with remarkable selectivity and potency. Employing a combination of nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) kinetic experiments, comprehensive enzymatic characterizations, and X-ray crystallography, we dissect the intricate details of the DFMO-HDAC6 interaction dynamics. More specifically, we find that the chemical structure of a DMFO and the binding mode of its difluoroacetylhydrazide derivative are crucial in determining the predominant hydrolysis mechanism. Our findings provide additional insights into two different mechanisms of DFMO hydrolysis, thus contributing to a better understanding of the HDAC6 inhibition by oxadiazoles in disease modulation and therapeutic intervention. Full article

(This article belongs to the Special Issue Mechanism of Enzyme Catalysis: When Structure Meets Function)

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14 pages, 4056 KiB

Open AccessArticle

Nontraditional Roles of Magnesium Ions in Modulating Sav2152: Insight from a Haloacid Dehalogenase-like Superfamily Phosphatase from Staphylococcus aureus

by Jaeseok Bang, Jaehui Park, Sung-Hee Lee, Jinhwa Jang, Junwoo Hwang, Otabek Kamarov, Hae-Joon Park, Soo-Jae Lee, Min-Duk Seo, Hyung-Sik Won, Seung-Hyeon Seok and Ji-Hun Kim

Int. J. Mol. Sci. 2024, 25(9), 5021; https://doi.org/10.3390/ijms25095021 - 4 May 2024

Viewed by 931

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) infection has rapidly spread through various routes. A genomic analysis of clinical MRSA samples revealed an unknown protein, Sav2152, predicted to be a haloacid dehalogenase (HAD)-like hydrolase, making it a potential candidate for a novel drug target. In this study, we determined the crystal structure of Sav2152, which consists of a C2-type cap domain and a core domain. The core domain contains four motifs involved in phosphatase activity that depend on the presence of Mg²⁺ ions. Specifically, residues D10, D12, and D233, which closely correspond to key residues in structurally homolog proteins, are responsible for binding to the metal ion and are known to play critical roles in phosphatase activity. Our findings indicate that the Mg²⁺ ion known to stabilize local regions surrounding it, however, paradoxically, destabilizes the local region. Through mutant screening, we identified D10 and D12 as crucial residues for metal binding and maintaining structural stability via various uncharacterized intra-protein interactions, respectively. Substituting D10 with Ala effectively prevents the interaction with Mg²⁺ ions. The mutation of D12 disrupts important structural associations mediated by D12, leading to a decrease in the stability of Sav2152 and an enhancement in binding affinity to Mg²⁺ ions. Additionally, our study revealed that D237 can replace D12 and retain phosphatase activity. In summary, our work uncovers the novel role of metal ions in HAD-like phosphatase activity. Full article

(This article belongs to the Special Issue Mechanism of Enzyme Catalysis: When Structure Meets Function)

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Review

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24 pages, 5865 KiB

Open AccessReview

4-Hydroxyphenylacetate 3-Hydroxylase (4HPA3H): A Vigorous Monooxygenase for Versatile O-Hydroxylation Applications in the Biosynthesis of Phenolic Derivatives

by Ping Sun, Shuping Xu, Yuan Tian, Pengcheng Chen, Dan Wu and Pu Zheng

Int. J. Mol. Sci. 2024, 25(2), 1222; https://doi.org/10.3390/ijms25021222 - 19 Jan 2024

Cited by 1 | Viewed by 1116

Abstract

4-Hydroxyphenylacetate 3-hydroxylase (4HPA3H) is a long-known class of two-component flavin-dependent monooxygenases from bacteria, including an oxygenase component (EC 1.14.14.9) and a reductase component (EC 1.5.1.36), with the latter being accountable for delivering the cofactor (reduced flavin) essential for o-hydroxylation. 4HPA3H has a broad substrate spectrum involved in key biological processes, including cellular catabolism, detoxification, and the biosynthesis of bioactive molecules. Additionally, it specifically hydroxylates the o-position of the C4 position of the benzene ring in phenolic compounds, generating high-value polyhydroxyphenols. As a non-P450 o-hydroxylase, 4HPA3H offers a viable alternative for the de novo synthesis of valuable natural products. The enzyme holds the potential to replace plant-derived P450s in the o-hydroxylation of plant polyphenols, addressing the current significant challenge in engineering specific microbial strains with P450s. This review summarizes the source distribution, structural properties, and mechanism of 4HPA3Hs and their application in the biosynthesis of natural products in recent years. The potential industrial applications and prospects of 4HPA3H biocatalysts are also presented. Full article

(This article belongs to the Special Issue Mechanism of Enzyme Catalysis: When Structure Meets Function)

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