Discovery of Bacterial Deaminases That Convert 5-Fluoroisocytosine Into 5-Fluorouracil

Abstract

Cytosine is one of the four letters of a standard genetic code, found both in DNA and in RNA. This heterocyclic base can be converted into uracil upon the action of the well-known cytosine deaminase. Isocytosine (2-aminouracil) is an isomer of cytosine, yet the enzymes that could convert it into uracil were previously mainly overlooked. In order to search for the isocytosine deaminases we used a selection strategy that is based on uracil auxotrophy and the metagenomic libraries, which provide a random pool of genes from uncultivated soil bacteria. Several genes that encode isocytosine deaminases were found and two respective recombinant proteins were purified. It was established that both novel deaminases do not use cytosine as a substrate. Instead, these enzymes are able to convert not only isocytosine into uracil, but also 5-fluoroisocytosine into 5-fluorouracil. Our findings suggest that novel isocytosine deaminases have a potential to be efficiently used in targeted cancer therapy instead of the classical cytosine deaminases. Use of isocytosine instead of cytosine would produce fewer side effects since deaminases produced by the commensal E. coli gut flora are ten times less efficient in degrading isocytosine than cytosine. In addition, there are no known homologs of isocytosine deaminases in human cells that would induce the toxicity when 5-fluoroisocytosine would be used as a prodrug.

Isocytosine deaminase Vcz as a novel tool for the prodrug cancer therapy

“…It has been proposed that the 5-FC toxicity might be due to production of 5-FU by the intestinal flora [22]. Recently, by screening of the environmental metagenomic libraries, a novel isocytosine deaminase (ICD), termed Vcz, which specifically converts 5-fluoroisocytosine (5-FIC) into 5-fluorouracil (5-FU), has been identified [23]. We predict that the gut flora would not metabolize 5-FIC as extensively as it does in the case of 5-FC.…”

Identification of a 2′-O-Methyluridine Nucleoside Hydrolase Using the Metagenomic Libraries

“…Enzymatic synthesis of methylated nucleosides has only recently been achieved [ 28 , 29 ], which means that the RK9NH enzyme is the gateway to a new and promising field of biocatalysis. In addition, since 5-fluoro-2′- O -methyluridine is a good substrate of RK9NH hydrolase, a novel prodrug/enzyme combination might be considered as an addition to the existing systems generating 5-fluorouracil, a well-known anticancer compound [ 46 , 47 , 48 ].…”

A Rapid Method for the Selection of Amidohydrolases from Metagenomic Libraries by Applying Synthetic Nucleosides and a Uridine Auxotrophic Host

“…The hydrolytic activity of the recombinant enzymes was assayed qualitatively by testing 29 different substrates ( Figure 4A): N 4 -acylated (2'-deoxy)cytidines and cytosines (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16); N 2 -acetylisocytosines (17); chromogenic substrates such as p-nitroacetanilide (18) and p-nitrobenzanilide (19); various p-nitrophenyl (pNP) esters (20)(21)(22)(23)(24); terephthalate derivatives bis(2-hydroxyethyl) terephthalate (26), dimethyl terephthalate (27), and monomethyl terephthalate (28); and the beta-lactam nitrocefin (29). Hydrolysis of chromogenic substrates (18)(19)(20)(21)(22)(23)(24)(25) was analysed spectrophotometrically at 405 nm, and that of other substrates was analysed spectrophotometrically at 240-320 nm and using TLC or HPLC-MS ( Figures S4-S10).…”

“…Keeping in mind that the functions of 30%-40% of the genes in genomes remain unknown, the detection of new biocatalysts in the sequenced genomes can be difficult if no significant homology with known enzymes is observed [6,7]. Metagenomics, the improvement of high-throughput screening methods, and the development of host expression systems for metagenome-derived genes, systems biology, and gene synthesis may together open the gateway to the useful information that hides in unexplored genetic resources, e.g., new enzymes with unique properties and novel scaffolds applicable for evolution in vitro [8][9][10][11][12].…”

“…Recently, it has been shown that a uridine auxotrophic strain of Escherichia coli can be successfully used for the identification of certain enzymes involved in the catabolism of modified heterocyclic bases or nucleosides [10,11,27] and for functional screening of esterases [12], when the appropriate uracil or uridine derivatives serve as substrates for the enzymes. Here, we present a method based on uridine auxotrophic E. coli, tailored especially for the selection of amidohydrolases from metagenomes.…”