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BMC Research Notes Mar 2021Non-haem iron(II)- and 2-oxoglutarate-dependent dioxygenases (i2OGdd), are a taxonomically and functionally diverse group of enzymes. The active site comprises ferrous...
Fe(2)OG: an integrated HMM profile-based web server to predict and analyze putative non-haem iron(II)- and 2-oxoglutarate-dependent dioxygenase function in protein sequences.
OBJECTIVE
Non-haem iron(II)- and 2-oxoglutarate-dependent dioxygenases (i2OGdd), are a taxonomically and functionally diverse group of enzymes. The active site comprises ferrous iron in a hexa-coordinated distorted octahedron with the apoenzyme, 2-oxoglutarate and a displaceable water molecule. Current information on novel i2OGdd members is sparse and relies on computationally-derived annotation schema. The dissimilar amino acid composition and variable active site geometry thereof, results in differing reaction chemistries amongst i2OGdd members. An additional need of researchers is a curated list of sequences with putative i2OGdd function which can be probed further for empirical data.
RESULTS
This work reports the implementation of [Formula: see text], a web server with dual functionality and an extension of previous work on i2OGdd enzymes [Formula: see text]. [Formula: see text], in this form is completely revised, updated (URL, scripts, repository) and will strengthen the knowledge base of investigators on i2OGdd biochemistry and function. [Formula: see text], utilizes the superior predictive propensity of HMM-profiles of laboratory validated i2OGdd members to predict probable active site geometries in user-defined protein sequences. [Formula: see text], also provides researchers with a pre-compiled list of analyzed and searchable i2OGdd-like sequences, many of which may be clinically relevant. [Formula: see text], is freely available ( http://204.152.217.16/Fe2OG.html ) and supersedes all previous versions, i.e., H2OGpred, DB2OG.
Topics: Amino Acid Sequence; Catalytic Domain; Dioxygenases; Ferrous Compounds; Ketoglutaric Acids
PubMed: 33648553
DOI: 10.1186/s13104-021-05477-z -
RSC Advances Feb 2021Recent studies have shown that inhibition of the hSIRT2 enzyme provides favorable effects in neurodegenerative diseases such as Alzheimer's disease. Prenylated xanthone...
Recent studies have shown that inhibition of the hSIRT2 enzyme provides favorable effects in neurodegenerative diseases such as Alzheimer's disease. Prenylated xanthone phytochemicals including α-mangostin, β-mangostin and γ-mangostin obtained from , a well-established tropical plant, have been shown experimentally to inhibit sirtuin enzymatic activity. However, the molecular mechanism of this sirtuin inhibition has not been reported. Using comprehensive integrated computational techniques, we provide molecular and timewise dynamical insights into the structural alterations capable of facilitating therapeutically beneficial effects of these phytochemicals at the catalytic core of the hSIRT2 enzyme. Findings revealed the enhanced conformational stability and compactness of the hSIRT2 catalytic core upon binding of γ-mangostin relative to the apoenzyme and better than α-mangostin and β-mangostin. Although thermodynamic calculations revealed favorable binding of all the phytochemicals to the hSIRT2 enzyme, the presence of only hydroxy functional groups on γ-mangostin facilitated the occurrence of additional hydrogen bonds involving Pro115, Phe119, Asn168 and His187 which are absent in α-mangostin- and β-mangostin-bound systems. Per-residue energy contributions showed that van der Waals and more importantly electrostatic interactions are involved in catalytic core stability with Phe96, Tyr104 and Phe235 notably contributing π-π stacking, π-π T shaped and π-sigma interactions. Cumulatively, our study revealed the structural alterations leading to inhibition of hSIRT2 catalysis and findings from this study could be significantly important for the future design and development of sirtuin inhibitors in the management of Alzheimer's disease.
PubMed: 35423339
DOI: 10.1039/d0ra10459k -
Journal of Molecular Evolution Apr 2021The RNA World is one of the most widely accepted hypotheses explaining the origin of the genetic system used by all organisms today. It proposes that the tripartite...
The RNA World is one of the most widely accepted hypotheses explaining the origin of the genetic system used by all organisms today. It proposes that the tripartite system of DNA, RNA, and proteins was preceded by one consisting solely of RNA, which both stored genetic information and performed the molecular functions encoded by that genetic information. Current research into a potential RNA World revolves around the catalytic properties of RNA-based enzymes, or ribozymes. Well before the discovery of ribozymes, Harold White proposed that evidence for a precursor RNA world could be found within modern proteins in the form of coenzymes, the majority of which contain nucleobases or nucleoside moieties, such as Coenzyme A and S-adenosyl methionine, or are themselves nucleotides, such as ATP and NADH (a dinucleotide). These coenzymes, White suggested, had been the catalytic active sites of ancient ribozymes, which transitioned to their current forms after the surrounding ribozyme scaffolds had been replaced by protein apoenzymes during the evolution of translation. Since its proposal four decades ago, this groundbreaking hypothesis has garnered support from several different research disciplines and motivated similar hypotheses about other classes of cofactors, most notably iron-sulfur cluster cofactors as remnants of the geochemical setting of the origin of life. Evidence from prebiotic geochemistry, ribozyme biochemistry, and evolutionary biology, increasingly supports these hypotheses. Certain coenzymes and cofactors may bridge modern biology with the past and can thus provide insights into the elusive and poorly-recorded period of the origin and early evolution of life.
Topics: Coenzymes; Evolution, Molecular; Nucleotides; Origin of Life; Proteins; RNA; RNA, Catalytic
PubMed: 33547911
DOI: 10.1007/s00239-020-09988-4 -
International Journal of Molecular... Jan 2021Ferroptosis has been described recently as an iron-dependent cell death driven by peroxidation of membrane lipids. It is involved in the pathogenesis of a number of... (Review)
Review
Ferroptosis has been described recently as an iron-dependent cell death driven by peroxidation of membrane lipids. It is involved in the pathogenesis of a number of diverse diseases. From the other side, the induction of ferroptosis can be used to kill tumor cells as a novel therapeutic approach. Because of the broad clinical relevance, a comprehensive understanding of the ferroptosis-controlling protein network is necessary. Noteworthy, several proteins from this network are flavoenzymes. This review is an attempt to present the ferroptosis-related flavoproteins in light of their involvement in anti-ferroptotic and pro-ferroptotic roles. When available, the data on the structural stability of mutants and cofactor-free apoenzymes are discussed. The stability of the flavoproteins could be an important component of the cellular death processes.
Topics: Animals; Ferroptosis; Flavoproteins; Humans; Iron; Protein Stability
PubMed: 33406703
DOI: 10.3390/ijms22010430 -
ChemMedChem Mar 2021The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2M ) to digest two of its translated long polypeptides to form a number of mature proteins that are...
The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2M ) to digest two of its translated long polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replicating in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1M ), we have designed and synthesized a series of SC2M inhibitors that contain β-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2M active-site cysteine C145. All inhibitors display high potency with K values at or below 100 nM. The most potent compound, MPI3, has as a K value of 8.3 nM. Crystallographic analyses of SC2M bound to seven inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549/ACE2 cells. Two inhibitors, MPI5 and MPI8, completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5 μM and A549/ACE2 cells at 0.16-0.31 μM. Their virus inhibition potency is much higher than that of some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2M inhibitors with ultra-high antiviral potency.
Topics: A549 Cells; Alanine; Animals; Antiviral Agents; Catalytic Domain; Chlorocebus aethiops; Coronavirus 3C Proteases; Cysteine; Cysteine Proteinase Inhibitors; Humans; Microbial Sensitivity Tests; Protein Binding; Pyrrolidinones; SARS-CoV-2; Vero Cells
PubMed: 33283984
DOI: 10.1002/cmdc.202000924 -
The Journal of General and Applied... Apr 2021Pseudomonas chlororaphis B23 yields nitrile hydratase (NHase) used for the production of 5-cyanovaleramide at the industrial level. Although the nhpC gene (known as...
Pseudomonas chlororaphis B23 yields nitrile hydratase (NHase) used for the production of 5-cyanovaleramide at the industrial level. Although the nhpC gene (known as P47K) located just downstream of the NHase structural genes (nhpAB) has been important for efficient NHase expression, the key role of nhpC remains poorly studied. Here, we purified two NHases expressed in the presence and absence of nhpC, respectively, and characterized them. The purified NHase expressed with nhpC proved to be an iron-containing holo-NHase, while the purified one expressed without nhpC was identified as an apo-NHase, which was iron-deficient. These findings indicated that nhpC would play a crucial role in the post-translational incorporation of iron into the NHase active site as a metal chaperone. In the overall amino acid sequence of NhpC, only the N-terminus exhibited similarities to the CobW protein involved in cobalamin biosynthesis, the UreG and HypB proteins essential for the metallocenter biosynthesis of urease and hydrogenase, respectively. NhpC contains a P-loop motif known as a nucleotide-binding site, and Lys23 and Thr24 are conserved in the P-loop motif in NhpC. Expression analysis of NHase formed in the presence of each mutant NhpC (i.e., K23A and T24A) resulted in immunodetectable production of a mutant NhpC and remarkable expression of NHase lacking the enzyme activity. These findings suggested that an intact P-loop containing Lys23 and Thr24 would be essential for the NhpC function in vivo for the post-translational metallocenter assembly of NHase.
Topics: Amino Acid Sequence; Bacterial Proteins; Binding Sites; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Hydro-Lyases; Iron; Mutagenesis, Site-Directed; Pseudomonas chlororaphis; Recombinant Proteins; Urease
PubMed: 33162426
DOI: 10.2323/jgam.2020.03.003 -
DNA Repair Sep 2020DNA polymerase β (Pol β) is an essential mammalian enzyme involved in the repair of DNA damage during the base excision repair (BER) pathway. In hopes of faithfully... (Review)
Review
DNA polymerase β (Pol β) is an essential mammalian enzyme involved in the repair of DNA damage during the base excision repair (BER) pathway. In hopes of faithfully restoring the coding potential to damaged DNA during BER, Pol β first uses a lyase activity to remove the 5'-deoxyribose phosphate moiety from a nicked BER intermediate, followed by a DNA synthesis activity to insert a nucleotide triphosphate into the resultant 1-nucleotide gapped DNA substrate. This DNA synthesis activity of Pol β has served as a model to characterize the molecular steps of the nucleotidyl transferase mechanism used by mammalian DNA polymerases during DNA synthesis. This is in part because Pol β has been extremely amenable to X-ray crystallography, with the first crystal structure of apoenzyme rat Pol β published in 1994 by Dr. Samuel Wilson and colleagues. Since this first structure, the Wilson lab and colleagues have published an astounding 267 structures of Pol β that represent different liganded states, conformations, variants, and reaction intermediates. While many labs have made significant contributions to our understanding of Pol β, the focus of this article is on the long history of the contributions from the Wilson lab. We have chosen to highlight select seminal Pol β structures with emphasis on the overarching contributions each structure has made to the field.
Topics: Animals; Biochemistry; Crystallography, X-Ray; DNA Polymerase beta; DNA Repair; DNA Replication; History, 20th Century; History, 21st Century; Humans; Mammals; Models, Molecular; Protein Conformation; Rats; Substrate Specificity
PubMed: 33087265
DOI: 10.1016/j.dnarep.2020.102928 -
ACS Chemical Biology Sep 2020Angucyclines are a structurally diverse class of actinobacterial natural products defined by their varied polycyclic ring systems, which display a wide range of...
Angucyclines are a structurally diverse class of actinobacterial natural products defined by their varied polycyclic ring systems, which display a wide range of biological activities. We recently discovered lugdunomycin (), a highly rearranged polyketide antibiotic derived from the angucycline backbone that is synthesized via several yet unexplained enzymatic reactions. Here, we show via , , and structural analysis that the promiscuous reductase LugOII catalyzes both a C6 and an unprecedented C1 ketoreduction. This then sets the stage for the subsequent C-ring cleavage that is key to the rearranged scaffolds of . The 1.1 Å structures of LugOII in complex with either ligand 8--Methylrabelomycin () or 8--Methyltetrangomycin () and of apoenzyme were resolved, which revealed a canonical Rossman fold and a remarkable conformational change during substrate capture and release. Mutational analysis uncovered key residues for substrate access, position, and catalysis as well as specific determinants that control its dual functionality. The insights obtained in this work hold promise for the discovery and engineering of other promiscuous reductases that may be harnessed for the generation of novel biocatalysts for chemoenzymatic applications.
Topics: Alcohol Oxidoreductases; Anti-Bacterial Agents; Bacterial Proteins; Biocatalysis; Catalytic Domain; Crystallography, X-Ray; Mutagenesis, Site-Directed; Mutation; Oxidation-Reduction; Polyketides; Protein Binding; Streptomyces; Substrate Specificity
PubMed: 32840360
DOI: 10.1021/acschembio.0c00564 -
International Journal of Molecular... Aug 2020Hydrogenases are complex metalloenzymes, showing tremendous potential as H-converting redox catalysts for application in light-driven H production, enzymatic fuel cells... (Review)
Review
Hydrogenases are complex metalloenzymes, showing tremendous potential as H-converting redox catalysts for application in light-driven H production, enzymatic fuel cells and H-driven cofactor regeneration. They catalyze the reversible oxidation of hydrogen into protons and electrons. The apo-enzymes are not active unless they are modified by a complicated post-translational maturation process that is responsible for the assembly and incorporation of the complex metal center. The catalytic center is usually easily inactivated by oxidation, and the separation and purification of the active protein is challenging. The understanding of the catalytic mechanisms progresses slowly, since the purification of the enzymes from their native hosts is often difficult, and in some case impossible. Over the past decades, only a limited number of studies report the homologous or heterologous production of high yields of hydrogenase. In this review, we emphasize recent discoveries that have greatly improved our understanding of microbial hydrogenases. We compare various heterologous hydrogenase production systems as well as in vitro hydrogenase maturation systems and discuss their perspectives for enhanced biohydrogen production. Additionally, activities of hydrogenases isolated from either recombinant organisms or in vivo/in vitro maturation approaches were systematically compared, and future perspectives for this research area are discussed.
Topics: Bacterial Proteins; Hydrogenase; Industrial Microbiology; Iron-Sulfur Proteins; Protein Engineering
PubMed: 32824336
DOI: 10.3390/ijms21165890 -
BioRxiv : the Preprint Server For... Jul 2020The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2M ) to digest two of its translated polypeptides to form a number of mature proteins that are...
The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2M ) to digest two of its translated polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replication in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1M ), we have designed and synthesized a series of SC2M inhibitors that contain β-( -2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2M active site cysteine C145. All inhibitors display high potency with IC values at or below 100 nM. The most potent compound MPI3 has as an IC value as 8.5 nM. Crystallographic analyses of SC2M bound to 7 inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549 cells. Two inhibitors MP5 and MPI8 completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5 μM and A549 cells at 0.16-0.31 μM. Their virus inhibition potency is much higher than some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2M inhibitors with extreme potency. Due to the urgent matter of the COVID-19 pandemic, MPI5 and MPI8 may be quickly advanced to preclinical and clinical tests for COVID-19.
PubMed: 32766582
DOI: 10.1101/2020.07.28.223784