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Life
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Current Approaches in Molecular Enzymology
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Current Approaches in Molecular Enzymology

A special issue of Life (ISSN 2075-1729). This special issue belongs to the section "Proteins and Proteomics".

Deadline for manuscript submissions: closed (20 October 2021) | Viewed by 36423

Special Issue Editor

Dr. Attila Ambrus

E-Mail Website
Guest Editor
Department of Biochemistry and Molecular Biology, Semmelweis University, 1094 Budapest, Hungary
Interests: lipoamide dehydrogenase deficiency; α-ketoglutarate dehydrogenase complex; protein structure; multienzyme complexes; NMR spectroscopy; X-ray crystallography; cryo-EM; molecular enzymology

Special Issue Information

Dear Colleagues,

As our technological repertoire advances, we are capable of digging deeper and deeper into the atomic level structures and mechanisms to dissect molecular pathogeneses. Beyond intellectual interest, the revealed pieces of information are also very relevant in the development of novel and increasingly specific clinical intervention strategies. The applied state-of-the-art biochemical, biophysical, structural, bioinformatic and molecular biology approaches resuscitated and revolutionized molecular enzymology research. All these novel tools in research, together with the completion of the Human Genome Project, also help reinvent thought to be known molecular targets. Several enzymes prove now to display moonlighting functions, whose modulation are reported to lead to auxiliary pathogeneses in disease. Targeting even a single element in a complex pathomechanism might already ameliorate clinical symptoms and improve survival rate.

The motivation behind this Special Issue is to present via examples various up-to-date biomolecular techniques and scientific approaches that accelerate the elucidation of intricate molecular details in the course of enzymology research today.

Dr. Attila Ambrus
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Life is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2600 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • molecular enzymology 
  • molecular pathomechanism 
  • auxiliary and complex pathogeneses 
  • enzyme dysfuntion 
  • moonlighting functions 
  • pathogenic molecular structures 
  • disease-causing variants 
  • methods for molecular pathology research

Published Papers (10 papers)

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Research

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14 pages, 1110 KiB  
Article
Correlation of GAA Genotype and Acid-α-Glucosidase Enzyme Activity in Hungarian Patients with Pompe Disease
by Aniko Gal, Zoltán Grosz, Beata Borsos, Ildikó Szatmari, Agnes Sebők, Laszló Jávor, Veronika Harmath, Katalin Szakszon, Livia Dezsi, Eniko Balku, Zita Jobbagy, Agnes Herczegfalvi, Zsuzsanna Almássy, Levente Kerényi and Maria Judit Molnar
Life 2021, 11(6), 507; https://doi.org/10.3390/life11060507 - 31 May 2021
Cited by 1 | Viewed by 3202
Abstract
Pompe disease is caused by the accumulation of glycogen in the lysosomes due to a deficiency of the lysosomal acid-α-glucosidase (GAA) enzyme. Depending on residual enzyme activity, the disease manifests two distinct phenotypes. In this study, we assess an enzymatic and genetic analysis [...] Read more.
Pompe disease is caused by the accumulation of glycogen in the lysosomes due to a deficiency of the lysosomal acid-α-glucosidase (GAA) enzyme. Depending on residual enzyme activity, the disease manifests two distinct phenotypes. In this study, we assess an enzymatic and genetic analysis of Hungarian patients with Pompe disease. Twenty-four patients diagnosed with Pompe disease were included. Enzyme activity of acid-α-glucosidase was measured by mass spectrometry. Sanger sequencing and an MLPA of the GAA gene were performed in all patients. Twenty (83.33%) patients were classified as having late-onset Pompe disease and four (16.66%) had infantile-onset Pompe disease. Fifteen different pathogenic GAA variants were detected. The most common finding was the c.-32-13 T > G splice site alteration. Comparing the α-glucosidase enzyme activity of homozygous cases to the compound heterozygous cases of the c.-32-13 T > G disease-causing variant, the mean GAA activity in homozygous cases was significantly higher. The lowest enzyme activity was found in cases where the c.-32-13 T > G variant was not present. The localization of the identified sequence variations in regions encoding the crucial protein domains of GAA correlates with severe effects on enzyme activity. A better understanding of the impact of pathogenic gene variations may help earlier initiation of enzyme replacement therapy (ERT) if subtle symptoms occur. Further information on the effect of GAA gene variation on the efficacy of treatment and the extent of immune response to ERT would be of importance for optimal disease management and designing effective treatment plans. Full article
(This article belongs to the Special Issue Current Approaches in Molecular Enzymology)
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9 pages, 572 KiB  
Article
The Role of the Rare Variants in the Genes Encoding the Alpha-Ketoglutarate Dehydrogenase in Alzheimer’s Disease
by Dora Csaban, Klara Pentelenyi, Renata Toth-Bencsik, Anett Illes, Zoltan Grosz, Andras Gezsi and Maria Judit Molnar
Life 2021, 11(4), 321; https://doi.org/10.3390/life11040321 - 6 Apr 2021
Cited by 4 | Viewed by 2164
Abstract
There is increasing evidence that several mitochondrial abnormalities are present in the brains of patients with Alzheimer’s disease (AD). Decreased alpha-ketoglutarate dehydrogenase complex (αKGDHc) activity was identified in some patients with AD. The αKGDHc is a key enzyme in the Krebs cycle. This [...] Read more.
There is increasing evidence that several mitochondrial abnormalities are present in the brains of patients with Alzheimer’s disease (AD). Decreased alpha-ketoglutarate dehydrogenase complex (αKGDHc) activity was identified in some patients with AD. The αKGDHc is a key enzyme in the Krebs cycle. This enzyme is very sensitive to the harmful effect of reactive oxygen species, which gives them a critical role in the Alzheimer and mitochondrial disease research area. Previously, several genetic risk factors were described in association with AD. Our aim was to analyze the associations of rare damaging variants in the genes encoding αKGDHc subunits and AD. The three genes (OGDH, DLST, DLD) encoding αKGDHc subunits were sequenced from different brain regions of 11 patients with histologically confirmed AD and the blood of further 35 AD patients. As a control group, we screened 134 persons with whole-exome sequencing. In all subunits, a one–one rare variant was identified with unknown significance based on American College of Medical Genetics and Genomics (ACMG) classification. Based on the literature research and our experience, R263H mutation in the DLD gene seems likely to be pathogenic. In the different cerebral areas, the αKGDHc mutational profile was the same, indicating the presence of germline variants. We hypothesize that the heterozygous missense R263H in the DLD gene may have a role in AD as a mild genetic risk factor. Full article
(This article belongs to the Special Issue Current Approaches in Molecular Enzymology)
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19 pages, 3744 KiB  
Article
RNase A Domain-Swapped Dimers Produced Through Different Methods: Structure–Catalytic Properties and Antitumor Activity
by Riccardo Montioli, Rachele Campagnari, Sabrina Fasoli, Andrea Fagagnini, Andra Caloiu, Marcello Smania, Marta Menegazzi and Giovanni Gotte
Life 2021, 11(2), 168; https://doi.org/10.3390/life11020168 - 21 Feb 2021
Cited by 9 | Viewed by 1996
Abstract
Upon oligomerization, RNase A can acquire important properties, such as cytotoxicity against leukemic cells. When lyophilized from 40% acetic acid solutions, the enzyme self-associates through the so-called three-dimensional domain swapping (3D-DS) mechanism involving both N- and/or C-terminals. The same species are formed if [...] Read more.
Upon oligomerization, RNase A can acquire important properties, such as cytotoxicity against leukemic cells. When lyophilized from 40% acetic acid solutions, the enzyme self-associates through the so-called three-dimensional domain swapping (3D-DS) mechanism involving both N- and/or C-terminals. The same species are formed if the enzyme is subjected to thermal incubation in various solvents, especially in 40% ethanol. We evaluated here if significant structural modifications might occur in RNase A N- or C-swapped dimers and/or in the residual monomer(s), as a function of the oligomerization protocol applied. We detected that the monomer activity vs. ss-RNA was partly affected by both protocols, although the protein does not suffer spectroscopic alterations. Instead, the two N-swapped dimers showed differences in the fluorescence emission spectra but almost identical enzymatic activities, while the C-swapped dimers displayed slightly different activities vs. both ss- or ds-RNA substrates together with not negligible fluorescence emission alterations within each other. Besides these results, we also discuss the reasons justifying the different relative enzymatic activities displayed by the N-dimers and C-dimers. Last, similarly with data previously registered in a mouse model, we found that both dimeric species significantly decrease human melanoma A375 cell viability, while only N-dimers reduce human melanoma MeWo cell growth. Full article
(This article belongs to the Special Issue Current Approaches in Molecular Enzymology)
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21 pages, 4714 KiB  
Article
Biochemical Characterization, Specificity and Inhibition Studies of HTLV-1, HTLV-2, and HTLV-3 Proteases
by Norbert Kassay, János András Mótyán, Krisztina Matúz, Mária Golda and József Tőzsér
Life 2021, 11(2), 127; https://doi.org/10.3390/life11020127 - 6 Feb 2021
Cited by 8 | Viewed by 3021
Abstract
The human T-lymphotropic viruses (HTLVs) are causative agents of severe diseases including adult T-cell leukemia. Similar to human immunodeficiency viruses (HIVs), the viral protease (PR) plays a crucial role in the viral life-cycle via the processing of the viral polyproteins. Thus, it is [...] Read more.
The human T-lymphotropic viruses (HTLVs) are causative agents of severe diseases including adult T-cell leukemia. Similar to human immunodeficiency viruses (HIVs), the viral protease (PR) plays a crucial role in the viral life-cycle via the processing of the viral polyproteins. Thus, it is a potential target of anti-retroviral therapies. In this study, we performed in vitro comparative analysis of human T-cell leukemia virus type 1, 2, and 3 (HTLV-1, -2, and -3) proteases. Amino acid preferences of S4 to S1′ subsites were studied by using a series of synthetic oligopeptide substrates representing the natural and modified cleavage site sequences of the proteases. Biochemical characteristics of the different PRs were also determined, including catalytic efficiencies and dependence of activity on pH, temperature, and ionic strength. We investigated the effects of different HIV-1 PR inhibitors (atazanavir, darunavir, DMP-323, indinavir, ritonavir, and saquinavir) on enzyme activities, and inhibitory potentials of IB-268 and IB-269 inhibitors that were previously designed against HTLV-1 PR. Comparative biochemical analysis of HTLV-1, -2, and -3 PRs may help understand the characteristic similarities and differences between these enzymes in order to estimate the potential of the appearance of drug-resistance against specific HTLV-1 PR inhibitors. Full article
(This article belongs to the Special Issue Current Approaches in Molecular Enzymology)
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Review

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14 pages, 839 KiB  
Review
Advanced CRISPR-Cas Effector Enzyme-Based Diagnostics for Infectious Diseases, Including COVID-19
by Sangha Kwon and Ha Youn Shin
Life 2021, 11(12), 1356; https://doi.org/10.3390/life11121356 - 7 Dec 2021
Cited by 10 | Viewed by 5219
Abstract
Rapid and precise diagnostic tests can prevent the spread of diseases, including worldwide pandemics. Current commonly used diagnostic methods include nucleic-acid-amplification-based detection methods and immunoassays. These techniques, however, have several drawbacks in diagnosis time, accuracy, and cost. Nucleic acid amplification methods are sensitive [...] Read more.
Rapid and precise diagnostic tests can prevent the spread of diseases, including worldwide pandemics. Current commonly used diagnostic methods include nucleic-acid-amplification-based detection methods and immunoassays. These techniques, however, have several drawbacks in diagnosis time, accuracy, and cost. Nucleic acid amplification methods are sensitive but time-consuming, whereas immunoassays are more rapid but relatively insensitive. Recently developed CRISPR-based nucleic acid detection methods have been found to compensate for these limitations. In particular, the unique collateral enzymatic activities of Cas12 and Cas13 have dramatically reduced the diagnosis times and costs, while improving diagnostic accuracy and sensitivity. This review provides a comprehensive description of the distinct enzymatic features of Cas12 and Cas13 and their applications in the development of molecular diagnostic platforms for pathogen detection. Moreover, it describes the current utilization of CRISPR-Cas-based diagnostic techniques to identify SARS-CoV-2 infection, as well as recent progress in the development of CRISPR-Cas-based detection strategies for various infectious diseases. These findings provide insights into designing effective molecular diagnostic platforms for potential pandemics. Full article
(This article belongs to the Special Issue Current Approaches in Molecular Enzymology)
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19 pages, 757 KiB  
Review
TAT for Enzyme/Protein Delivery to Restore or Destroy Cell Activity in Human Diseases
by Michal Lichtenstein, Samar Zabit, Noa Hauser, Sarah Farouz, Orly Melloul, Joud Hirbawi and Haya Lorberboum-Galski
Life 2021, 11(9), 924; https://doi.org/10.3390/life11090924 - 6 Sep 2021
Cited by 8 | Viewed by 3010
Abstract
Much effort has been dedicated in the recent decades to find novel protein/enzyme-based therapies for human diseases, the major challenge of such therapies being the intracellular delivery and reaching sub-cellular organelles. One promising approach is the use of cell-penetrating peptides (CPPs) for delivering [...] Read more.
Much effort has been dedicated in the recent decades to find novel protein/enzyme-based therapies for human diseases, the major challenge of such therapies being the intracellular delivery and reaching sub-cellular organelles. One promising approach is the use of cell-penetrating peptides (CPPs) for delivering enzymes/proteins into cells. In this review, we describe the potential therapeutic usages of CPPs (mainly trans-activator of transcription protein, TAT) in enabling the uptake of biologically active proteins/enzymes needed in cases of protein/enzyme deficiency, concentrating on mitochondrial diseases and on the import of enzymes or peptides in order to destroy pathogenic cells, focusing on cancer cells. Full article
(This article belongs to the Special Issue Current Approaches in Molecular Enzymology)
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24 pages, 3646 KiB  
Review
Toward an Understanding of the Structural and Mechanistic Aspects of Protein-Protein Interactions in 2-Oxoacid Dehydrogenase Complexes
by Natalia S. Nemeria, Xu Zhang, Joao Leandro, Jieyu Zhou, Luying Yang, Sander M. Houten and Frank Jordan
Life 2021, 11(5), 407; https://doi.org/10.3390/life11050407 - 29 Apr 2021
Cited by 12 | Viewed by 3397
Abstract
The 2-oxoglutarate dehydrogenase complex (OGDHc) is a key enzyme in the tricarboxylic acid (TCA) cycle and represents one of the major regulators of mitochondrial metabolism through NADH and reactive oxygen species levels. The OGDHc impacts cell metabolic and cell signaling pathways through the [...] Read more.
The 2-oxoglutarate dehydrogenase complex (OGDHc) is a key enzyme in the tricarboxylic acid (TCA) cycle and represents one of the major regulators of mitochondrial metabolism through NADH and reactive oxygen species levels. The OGDHc impacts cell metabolic and cell signaling pathways through the coupling of 2-oxoglutarate metabolism to gene transcription related to tumor cell proliferation and aging. DHTKD1 is a gene encoding 2-oxoadipate dehydrogenase (E1a), which functions in the L-lysine degradation pathway. The potentially damaging variants in DHTKD1 have been associated to the (neuro) pathogenesis of several diseases. Evidence was obtained for the formation of a hybrid complex between the OGDHc and E1a, suggesting a potential cross talk between the two metabolic pathways and raising fundamental questions about their assembly. Here we reviewed the recent findings and advances in understanding of protein-protein interactions in OGDHc and 2-oxoadipate dehydrogenase complex (OADHc), an understanding that will create a scaffold to help design approaches to mitigate the effects of diseases associated with dysfunction of the TCA cycle or lysine degradation. A combination of biochemical, biophysical and structural approaches such as chemical cross-linking MS and cryo-EM appears particularly promising to provide vital information for the assembly of 2-oxoacid dehydrogenase complexes, their function and regulation. Full article
(This article belongs to the Special Issue Current Approaches in Molecular Enzymology)
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23 pages, 8188 KiB  
Review
Two Decades of Evolution of Our Understanding of the Transient Receptor Potential Melastatin 2 (TRPM2) Cation Channel
by Andras Szollosi
Life 2021, 11(5), 397; https://doi.org/10.3390/life11050397 - 27 Apr 2021
Cited by 11 | Viewed by 2980
Abstract
The transient receptor potential melastatin (TRPM) family belongs to the superfamily of TRP ion channels. It consists of eight family members that are involved in a plethora of cellular functions. TRPM2 is a homotetrameric Ca2+-permeable cation channel activated upon oxidative stress [...] Read more.
The transient receptor potential melastatin (TRPM) family belongs to the superfamily of TRP ion channels. It consists of eight family members that are involved in a plethora of cellular functions. TRPM2 is a homotetrameric Ca2+-permeable cation channel activated upon oxidative stress and is important, among others, for body heat control, immune cell activation and insulin secretion. Invertebrate TRPM2 proteins are channel enzymes; they hydrolyze the activating ligand, ADP-ribose, which is likely important for functional regulation. Since its cloning in 1998, the understanding of the biophysical properties of the channel has greatly advanced due to a vast number of structure–function studies. The physiological regulators of the channel have been identified and characterized in cell-free systems. In the wake of the recent structural biochemistry revolution, several TRPM2 cryo-EM structures have been published. These structures have helped to understand the general features of the channel, but at the same time have revealed unexplained mechanistic differences among channel orthologues. The present review aims at depicting the major research lines in TRPM2 structure-function. It discusses biophysical properties of the pore and the mode of action of direct channel effectors, and interprets these functional properties on the basis of recent three-dimensional structural models. Full article
(This article belongs to the Special Issue Current Approaches in Molecular Enzymology)
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38 pages, 6064 KiB  
Review
Proton Bridging in Catalysis by and Inhibition of Serine Proteases of the Blood Cascade System
by Ildiko M Kovach
Life 2021, 11(5), 396; https://doi.org/10.3390/life11050396 - 27 Apr 2021
Cited by 2 | Viewed by 2764
Abstract
Inquiries into the participation of short hydrogen bonds in stabilizing transition states and intermediate states in the thrombin, factor Xa, plasmin and activated protein C–catalyzed reactions revealed that specific binding of effectors at Sn, n = 1–4 and S’n, [...] Read more.
Inquiries into the participation of short hydrogen bonds in stabilizing transition states and intermediate states in the thrombin, factor Xa, plasmin and activated protein C–catalyzed reactions revealed that specific binding of effectors at Sn, n = 1–4 and S’n, n = 1–3 and at remote exosites elicit complex patterns of hydrogen bonding and involve water networks. The methods employed that yielded these discoveries include; (1) kinetics, especially partial or full kinetic deuterium solvent isotope effects with short cognate substrates and also with the natural substrates, (2) kinetic and structural probes, particularly low-field high-resolution nuclear magnetic resonance (1H NMR), of mechanism-based inhibitors and substrate-mimic peptide inhibitors. Short hydrogen bonds form at the transition states of the catalytic reactions at the active site of the enzymes as they do with mechanism-based covalent inhibitors of thrombin. The emergence of short hydrogen bonds at the binding interface of effectors and thrombin at remote exosites has recently gained recognition. Herein, I describe our contribution, a confirmation of this discovery, by low-field 1H NMR. The principal conclusion of this review is that proton sharing at distances below the sum of van der Waals radii of the hydrogen and both donor and acceptor atoms contribute to the remarkable catalytic prowess of serine proteases of the blood clotting system and other enzymes that employ acid-base catalysis. Proton bridges also play a role in tight binding in proteins and at exosites, i.e., allosteric sites, of enzymes. Full article
(This article belongs to the Special Issue Current Approaches in Molecular Enzymology)
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19 pages, 2136 KiB  
Review
Hydrogen-Deuterium Exchange Mass Spectrometry: A Novel Structural Biology Approach to Structure, Dynamics and Interactions of Proteins and Their Complexes
by Oliver Ozohanics and Attila Ambrus
Life 2020, 10(11), 286; https://doi.org/10.3390/life10110286 - 15 Nov 2020
Cited by 25 | Viewed by 7315
Abstract
Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) is a rapidly evolving technique for analyzing structural features and dynamic properties of proteins. It may stand alone or serve as a complementary method to cryo-electron-microscopy (EM) or other structural biology approaches. HDX-MS is capable of providing information [...] Read more.
Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) is a rapidly evolving technique for analyzing structural features and dynamic properties of proteins. It may stand alone or serve as a complementary method to cryo-electron-microscopy (EM) or other structural biology approaches. HDX-MS is capable of providing information on individual proteins as well as large protein complexes. Owing to recent methodological advancements and improving availability of instrumentation, HDX-MS is becoming a routine technique for some applications. When dealing with samples of low to medium complexity and sizes of less than 150 kDa, conformation and ligand interaction analyses by HDX-MS are already almost routine applications. This is also well supported by the rapid evolution of the computational (software) background that facilitates the analysis of the obtained experimental data. HDX-MS can cope at times with analytes that are difficult to tackle by any other approach. Large complexes like viral capsids as well as disordered proteins can also be analyzed by this method. HDX-MS has recently become an established tool in the drug discovery process and biopharmaceutical development, as it is now also capable of dissecting post-translational modifications and membrane proteins. This mini review provides the reader with an introduction to the technique and a brief overview of the most common applications. Furthermore, the most challenging likely applications, the analyses of glycosylated and membrane proteins, are also highlighted. Full article
(This article belongs to the Special Issue Current Approaches in Molecular Enzymology)
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