skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


Title: Computational investigations of selected enzymes from two iron and α-ketoglutarate-dependent families
DNA alkylation is used as the key epigenetic mark in eukaryotes, however, most alkylation in DNA can result in deleterious effects. Therefore, this process needs to be tightly regulated. The enzymes of the AlkB and Ten-Eleven Translocation (TET) families are members of the Fe and alpha-ketoglutarate-dependent superfamily of enzymes that are tasked with dealkylating DNA and RNA in cells. Members of these families span all species and are an integral part of transcriptional regulation. While both families catalyze oxidative dealkylation of various bases, each has specific preference for alkylated base type as well as distinct catalytic mechanisms. This perspective aims to provide an overview of computational work carried out to investigate several members of these enzyme families including AlkB, ALKB Homolog 2, ALKB Homolog 3 and Ten-Eleven Translocate 2. Insights into structural details, mutagenesis studies, reaction path analysis, electronic structure features in the active site, and substrate preferences are presented and discussed.  more » « less
Award ID(s):
1856162 2217856
PAR ID:
10325303
Author(s) / Creator(s):
; ; ;
Date Published:
Journal Name:
Physical Chemistry Chemical Physics
Volume:
23
Issue:
39
ISSN:
1463-9076
Page Range / eLocation ID:
22227 to 22240
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; ; ; ; ; ; et al (, Research Square)
    Abstract DNA methylation at cytosine bases of eukaryotic DNA (5-methylcytosine, 5mC) is a heritable epigenetic mark that can regulate gene expression in health and disease. Enzymes that metabolize 5mC have been well-characterized, yet the discovery of endogenously produced signaling molecules that regulate DNA methyl-modifying machinery have not been described. Herein, we report that the free radical signaling molecule nitric oxide (NO) can directly inhibit the Fe(II)/2-OG-dependent DNA demethylases ten-eleven translocation (TET) and human AlkB homolog 2 (ALKBH2). Physiologic NO concentrations reversibly inhibited TET and ALKBH2 demethylase activity by binding to the mononuclear non-heme iron atom which formed a dinitrosyliron complex (DNIC) preventing cosubstrates (2-OG and O2) from binding. In cancer cells treated with exogenous NO, or cells endogenously synthesizing NO, there was a global increase in 5mC and 5-hydroxymethylcytosine (5hmC) in DNA, the substrates for TET, that could not be attributed to increased DNA methyltransferase activity. 5mC was also elevated in NO-producing cell-line-derived mouse xenograft and patient-derived xenograft tumors. Genome-wide DNA methylome analysis of cells chronically treated with NO (10 days) demonstrated enrichment of 5mC and 5hmC at gene-regulatory loci which correlated to changes in the expression of NO-regulated tumor-associated genes. Regulation of DNA methylation is distinctly different from canonical NO signaling and represents a novel epigenetic role for NO. 
    more » « less
  2. ; ; (, Journal of Biological Chemistry)
    AlkB is a bacterial Fe(II)– and 2-oxoglutarate–dependent dioxygenase that repairs a wide range of alkylated nucleobases in DNA and RNA as part of the adaptive response to exogenous nucleic acid–alkylating agents. Although there has been longstanding interest in the structure and specificity of Escherichia coli AlkB and its homologs, difficulties in assaying their repair activities have limited our understanding of their substrate specificities and kinetic mechanisms. Here, we used quantitative kinetic approaches to determine the transient kinetics of recognition and repair of alkylated DNA by AlkB. These experiments revealed that AlkB is a much faster alkylation repair enzyme than previously reported and that it is significantly faster than DNA repair glycosylases that recognize and excise some of the same base lesions. We observed that whereas 1, N 6 -ethenoadenine can be repaired by AlkB with similar efficiencies in both single- and double-stranded DNA, 1-methyladenine is preferentially repaired in single-stranded DNA. Our results lay the groundwork for future studies of AlkB and its human homologs ALKBH2 and ALKBH3. 
    more » « less
  3. ; ; ; ; ; ; (, Chemical Science)
    Ten-eleven translocation (TET) enzymes oxidize C–H bonds in 5-methylcytosine (5mC) to hydroxyl (5hmC), formyl (5fC) and carboxyl (5caC) intermediates en route to DNA demethylation. It has remained a challenge to study the function of a single oxidized product. We investigate whether alkyl groups other than methyl could be oxidized by TET proteins to generate a specific intermediate. We report here that TET2 oxidizes 5-ethylcytosine (5eC) only to 5-hydroxyethylcytosine (5heC). In biochemical assays, 5heC acts as a docking site for proteins implicated in transcription, imbuing this modification with potential gene regulatory activity. We observe that 5heC is resistant to downstream wild type hydrolases, but not to the engineered enzymes, thus establishing a unique tool to conditionally alter the stability of 5heC on DNA. Furthermore, we devised a chemical approach for orthogonal labeling of 5heC. Our work offers a platform for synthesis of novel 5-alkylcytosines, provides an approach to ‘tame’ TET activity, and identifies 5heC as an unnatural modification with a potential to control chromatin-dependent processes. 
    more » « less
  4. ; ; ; ; ; ; ; (, Proceedings of the National Academy of Sciences)
    We have developed Differential Specificity and Energy Landscape (DiSEL) analysis to comprehensively compare DNA–protein interactomes (DPIs) obtained by high-throughput experimental platforms and cutting edge computational methods. While high-affinity DNA binding sites are identified by most methods, DiSEL uncovered nuanced sequence preferences displayed by homologous transcription factors. Pairwise analysis of 726 DPIs uncovered homolog-specific differences at moderate- to low-affinity binding sites (submaximal sites). DiSEL analysis of variants of 41 transcription factors revealed that many disease-causing mutations result in allele-specific changes in binding site preferences. We focused on a set of highly homologous factors that have different biological roles but “read” DNA using identical amino acid side chains. Rather than direct readout, our results indicate that DNA noncontacting side chains allosterically contribute to sculpt distinct sequence preferences among closely related members of transcription factor families. 
    more » « less
  5. ; ; ; ; (, eLife)
    Billions of years ago, the Earth’s atmosphere had very little oxygen. It was only after some bacteria and early plants evolved to harness energy from sunlight that oxygen began to fill the Earth’s environment. Oxygen is highly reactive and can interfere with enzymes and other molecules that are essential to life. Organisms living at this point in history therefore had to adapt to survive in this new oxygen-rich world. An ancient family of enzymes known as ribonucleotide reductases are used by all free-living organisms and many viruses to repair and replicate their DNA. Because of their essential role in managing DNA, these enzymes have been around on Earth for billions of years. Understanding how they evolved could therefore shed light on how nature adapted to increasing oxygen levels and other environmental changes at the molecular level. One approach to study how proteins evolved is to use computational analysis to construct a phylogenetic tree. This reveals how existing members of a family are related to one another based on the chain of molecules (known as amino acids) that make up each protein. Despite having similar structures and all having the same function, ribonucleotide reductases have remarkably diverse sequences of amino acids. This makes it computationally very demanding to build a phylogenetic tree. To overcome this, Burnim, Spence, Xu et al. created a phylogenetic tree using structural information from a part of the enzyme that is relatively similar in many modern-day ribonucleotide reductases. The final result took seven continuous months on a supercomputer to generate, and includes over 6,000 members of the enzyme family. The phylogenetic tree revealed a new distinct group of ribonucleotide reductases that may explain how one adaptation to increasing levels of oxygen emerged in some family members, while another adaptation emerged in others. The approach used in this work also opens up a new way to study how other highly diverse enzymes and other protein families evolved, potentially revealing new insights about our planet’s past. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email