Phage-encoded ten-eleven translocation dioxygenase (TET) is active in C5-cytosine hypermodification in DNA

Burke, Evan J.; Rodda, Samuel S.; Lund, Sean R.; Sun, Zhiyi; Zeroka, Malcolm R.; O’Toole, Katherine H; Parker, Mackenzie J.; Doshi, Dharit S.; Guan, Chaowen; Lee, Yan‐Jiun; Dai, Nan; Hough, David M.; Shnider, Daria A.; Corrêa, Ivan R.; Weigele, Peter; Saleh, Lana

doi:10.1073/pnas.2026742118

Cited by 12 publications

(27 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The initial set of GT-containing contigs was curated from our previously published dataset of C5-MT and 5mYOX-containing pathways (18). Conserved pathway architecture (Fig.…”

Section: Initial Pathway Annotations and Synteny Predictionsmentioning

confidence: 99%

Virus-encoded glycosyltransferases hypermodify DNA with diverse glycans

Pyle,

Lund,

O’Toole

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

Enzymatic modification of DNA nucleobases can coordinate gene expression, protection from nucleases, or mutagenesis. We recently discovered a new clade of phage-specific cytosine methyltransferase (MT) and 5-methylpyrimidine dioxygenase (5mYOX,e.g.,TET) enzymes that produce 5-hydroxymethylcytosine (5hmC) as a precursor for additional post-replicative enzymatic hypermodifications on viral genomes. Here, we identify phage MT- and 5mYOX-dependent glycosyltransferase (GT) enzymes that catalyze linkage of diverse glycans directly onto 5hmC reactive nucleobase substrates. Using targeted bioinformatic mining of the phage metavirome databases, we discovered thousands of new biosynthetic gene clusters (BGCs) containing enzymes with predicted roles in cytosine sugar hypermodification. We developed a pathway reassembly platform for high-throughput functional screening of GT-containing BGCs, relying on the endogenousE. colimetabolome as a substrate pool. We successfully reconstituted a subset of phage BGCs and isolated novel and highly diverse sugar modifications appended to 5hmC, including mono-, di-, or tri-saccharide moieties comprised of hexose, N-acetylhexosamine or heptose sugars. Structural predictions and sugar product analyses suggest that phage GTs are related to host lipopolysaccharide, teichoic acid, and other small molecule biosynthesis enzymes and have been repurposed for DNA substrates. An expanded metagenomic search revealed hypermodification BGCs within gene neighborhoods containing phage structural proteins and putative genome defense systems. These findings enrich our knowledge of secondary modifications on DNA and the origins of corresponding sugar writer enzymes. Post-replicative cytosine hypermodification by virus-encoded GTs is discussed in the context of genome defense, DNA partitioning and virion assembly, and host-pathogen co-evolution.

show abstract

“…The initial set of GT-containing contigs was curated from our previously published dataset of C5-MT and 5mYOX-containing pathways (18). Conserved pathway architecture (Fig.…”

Section: Initial Pathway Annotations and Synteny Predictionsmentioning

confidence: 99%

Virus-encoded glycosyltransferases hypermodify DNA with diverse glycans

Pyle,

Lund,

O’Toole

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…O-Demethylation of codein(7) by O-demethylase (CODM) to produce morphine (8) 46. Ring closure in the biosynthesis of clavulanic acid, in which CS converts proclavaminic acid (9) into dihydroclavaminic acid(10) 47. Ring expansion of the penicillin N (11) thiazolidine ring to deacetoxycephalosporin C (12) catalyzed by deacetoxycephalosporin C synthase (DAOCS) 48.…”

mentioning

confidence: 99%

“…α- KGDs are widely distributed throughout living organisms and because of their extraordinary synthetic capabilities contribute significantly to many biological processes. For example, in mammals, α-KGDs take part in chromatin and DNA modifications, − tRNA demethylations/hydroxylations, − and post-translational modifications of proteins. − Moreover, within plants, bacteria, fungi, and mammals, these enzymes participate in biosynthetic pathways that generate numerous metabolites. ,, In chemical synthesis, α-KGDs are increasingly being sought because of their ability to regio- and stereospecifically activate C–H bonds in both simple and complex molecular structures. − In this context, α-KGDs are being explored in structure–activity relationship studies in medicinal chemistry, for the synthesis of antibiotics, − or for the chemoenzymatic synthesis of complex natural products. , Industrially, the hydroxylation of amino acids by α-KGDs, for example, in the context of producing hydroxylated lysine, proline, , and isoleucine derivatives, , has become a recognized manufacturing strategy for obtaining valuable small molecule building blocks.…”

mentioning

confidence: 99%

Engineering Fe(II)/α-Ketoglutarate-Dependent Halogenases and Desaturases

Papadopoulou

Buller

2022

Biochemistry

View full text Add to dashboard Cite

Fe(II)/α-ketoglutarate-dependent dioxygenases (α-KGDs) are widespread enzymes in aerobic biology and serve a remarkable array of biological functions, including roles in collagen biosynthesis, plant and animal development, transcriptional regulation, nucleic acid modification, and secondary metabolite biosynthesis. This functional diversity is reflected in the enzymes’ catalytic flexibility as α-KGDs can catalyze an intriguing set of synthetically valuable reactions, such as hydroxylations, halogenations, and desaturations, capturing the interest of scientists across disciplines. Mechanistically, all α-KGDs are understood to follow a similar activation pathway to generate a substrate radical, yet how individual members of the enzyme family direct this key intermediate toward the different reaction outcomes remains elusive, triggering structural, computational, spectroscopic, kinetic, and enzyme engineering studies. In this Perspective, we will highlight how first enzyme and substrate engineering examples suggest that the chemical reaction pathway within α-KGDs can be intentionally tailored using rational design principles. We will delineate the structural and mechanistic investigations of the reprogrammed enzymes and how they begin to inform about the enzymes’ structure–function relationships that determine chemoselectivity. Application of this knowledge in future enzyme and substrate engineering campaigns will lead to the development of powerful C–H activation catalysts for chemical synthesis.

show abstract

“…Followed by formation of hydroxymethyl pyrimidine nucleotides, which are utilized by DNA polymerase, replication and postreplicative modifications furnish installation of these moieties onto the DNA polymer (3-7). Burke et al (8) show that bacteriophages resort to C5-cytosine methyltransferase (C5-MT) and 5-methylcytosine dioxygenase ten-eleven translocation enzyme (TET) as an alternative mechanism to postreplicatively form hydroxymethylcytosine on DNA. The bacteriophage TET enables site-specific hydroxylation of 5-methylcytosine (5mC), installed by C5-MT, to produce 5-hydroxymethylcytosine (5hmC).…”

mentioning

confidence: 99%

“…Notably, the work carried out by Burke et al (8) demonstrates that bacteriophage TETs along with C5-MT and tailoring enzymes serve a role in phage DNA hypermodification. Using an Escherichia coli expression system and liquid chromatography-mass spectrometry/ mass spectrometry, Burke et al provide evidence that phage TETs perform a single oxidation event to convert 5mC to 5hmC, but no further oxidation as observed in eukaryotic TETs (Fig.…”

mentioning

confidence: 99%