Structural studies of yeast and bacterial cytosine deaminase: Evolution and implications for anticancer gene therapy

Cytosine deaminase (CD) catalyzes the deamination of cytosine, producing uracil. This enzyme is present in prokaryotes and fungi (but not multicellular eukaryotes) and is an important member of the pyrimidine salvage pathway in those organisms. The same enzyme also catalyzes the conversion of 5-fluorocytosine to 5-fluorouracil; this activity allows the formation of a cytotoxic chemotherapeutic agent from a non-cytotoxic precursor. The enzyme is of widespread interest both for antimicrobial drug design and for gene therapy applications against tumors. A comparison of Escherichia coli and Saccharomyces cerevisiae cytosine deaminases reveals significant differences between the bacterial and fungal enzymes, including their primary amino acid sequence, predicted molecular mass, quaternary structure, and relative substrate specificities and affinities, indicating that they are distinct and separately evolved enzymes. The structure of Escherichia coli CD has been determined in the presence and absence of a bound mechanism-based inhibitor. The enzyme forms an (αβ)₈ barrel structure with structural similarity to adenosine deaminase, a relationship that is undetectable at the sequence level, and no similarity to bacterial cytidine deaminase. The enzyme is packed into a hexameric assembly stabilized by a unique domain-swapping interaction between enzyme subunits. The active site is located in the mouth of the enzyme barrel and contains a bound iron ion that coordinates a hydroxyl nucleophile. Substrate binding involves a significant conformational change that sequesters the reaction complex from solvent. The structure of Saccharomyces cerevisiae CD has been determined in the presence and absence of a mechanism based inhibitor, at 1.14 Å and 1.43 Å resolution, respectively. The enzyme forms an α/β fold similar to bacterial cytidine deaminase, but with no similarity to the α/β barrel fold used by bacterial cytosine deaminase or mammalian adenosine deaminase. The structures observed for bacterial, fungal and mammalian nucleic acid deaminases represent an example of the parallel evolution of two unique protein folds to carry out the same reaction on a diverse array of substrates. Directed evolution of the bacterial cytosine deaminase has resulted in a mutant with enhanced sensitivity to 5-fluorocytosine, with implications for prodrug gene therapy applications.