Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.4.1 (cytosine deaminase)
747 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Pseudomonas sp. strain ADP metabolizes atrazine to cyanuric acid via three plasmid-encoded enzymes, AtzA, AtzB, and AtzC. The first enzyme, AtzA, catalyzes the hydrolytic dechlorination of atrazine, yielding hydroxyatrazine. The second enzyme, AtzB, catalyzes hydroxyatrazine deamidation, yielding N-isopropylammelide. In this study, the third gene in the atrazine catabolic pathway, atzC, was cloned from a Pseudomonas sp. strain ADP cosmid library as a 25-kb EcoRI DNA fragment in Escherichia coli. The atzC gene was further delimited by functional analysis following transposon Tn5 mutagenesis and subcloned as a 2.0-kb EcoRI-AvaI fragment. An E. coli strain containing this DNA fragment expressed N-isopropylammelide isopropylamino hydrolase activity, metabolizing N-isopropylammelide stoichiometrically to cyanuric acid and N-isopropylamine. The 2.0-kb DNA fragment was sequenced and found to contain a single open reading frame of 1,209 nucleotides, encoding a protein of 403 amino acids. AtzC showed modest sequence identity of 29 and 25%, respectively, to cytosine deaminase and dihydroorotase, both members of an amidohydrolase protein superfamily. The sequence of AtzC was compared to that of E. coli cytosine deaminase in the regions containing the five ligands to the catalytically important metal for the protein. Pairwise comparison of the 35 amino acids showed 61% sequence identity and 85% sequence similarity. AtzC is thus assigned to the amidohydrolase protein family that includes cytosine deaminase, urease, adenine deaminase, and phosphotriester hydrolase. Similar sequence comparisons of the most highly conserved regions indicated that the AtzA and AtzB proteins also belong to the same amidohydrolase family. Overall, the data suggest that AtzA, AtzB, and AtzC diverged from a common ancestor and, by random events, have been reconstituted onto an atrazine catabolic plasmid.
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PMID:AtzC is a new member of the amidohydrolase protein superfamily and is homologous to other atrazine-metabolizing enzymes. 942 5

Programmable nucleases are cutting edge genetic technology which edits targeted DNA sequences through generation of site-specific double-strand DNA breaks (DSBs). To improve the efficiency and precision of genetic modification, scientists have developed a single-base editing system (base editor) through combining of CRISPR/Cas9 system with cytosine deaminase. Compared with Cas9 system, this base editor can convert cytosine to thymine (C > T) at specific site more efficiently without inducing DSBs to avoid generation of indels. However, the base editor can only generate transition of pyrimidine but could not modify purines. Recently, Nature published a novel base editing system to convert adenine to guanine (ABEs, adenine base editors) through fusion of Cas9 nickase to a modified deaminase which is evolved through screening of random library based on tRNA adenine deaminase from E. coli. Here, we summarize the development of single-base editing tools and the latest research progress, especially the optimization process of ABEs, as well as the potential directions of the base editors.
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PMID:The "new favorite" of gene editing technology-single base editors. 2925 82

Recently developed DNA base editing methods enable the direct generation of desired point mutations in genomic DNA without generating any double-strand breaks^1-3, but the issue of off-target edits has limited the application of these methods. Although several previous studies have evaluated off-target mutations in genomic DNA^4-8, it is now clear that the deaminases that are integral to commonly used DNA base editors often bind to RNA^9-13. For example, the cytosine deaminase APOBEC1-which is used in cytosine base editors (CBEs)-targets both DNA and RNA¹², and the adenine deaminase TadA-which is used in adenine base editors (ABEs)-induces site-specific inosine formation on RNA^9,11. However, any potential RNA mutations caused by DNA base editors have not been evaluated. Adeno-associated viruses are the most common delivery system for gene therapies that involve DNA editing; these viruses can sustain long-term gene expression in vivo, so the extent of potential RNA mutations induced by DNA base editors is of great concern^14-16. Here we quantitatively evaluated RNA single nucleotide variations (SNVs) that were induced by CBEs or ABEs. Both the cytosine base editor BE3 and the adenine base editor ABE7.10 generated tens of thousands of off-target RNA SNVs. Subsequently, by engineering deaminases, we found that three CBE variants and one ABE variant showed a reduction in off-target RNA SNVs to the baseline while maintaining efficient DNA on-target activity. This study reveals a previously overlooked aspect of off-target effects in DNA editing and also demonstrates that such effects can be eliminated by engineering deaminases.
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PMID:Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis. 3118 67

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