Crystal Structure of Yeast Cytosine Deaminase

Abstract

Yeast cytosine deaminase is an attractive candidate for anticancer gene therapy because it catalyzes the deamination of the prodrug 5-fluorocytosine to form 5-fluorouracil. We report here the crystal structure of the enzyme in complex with the inhibitor 2-hydroxypyrimidine at 1.6-Å resolution. The protein forms a tightly packed dimer with an extensive interface of 1450 Å 2 per monomer. The inhibitor was converted into a hydrated adduct as a transition-state analog. The essential zinc ion is ligated by the 4-hydroxyl group of the inhibitor together with His 62 , Cys 91 , and Cys 94 from the protein. The enzyme shares similar active-site architecture to cytidine deaminases and an unusually high structural homology to 5-aminoimidazole-4-carboxamide-ribonucleotide transformylase and thereby may define a new superfamily. The unique C-terminal tail is involved in substrate specificity and also functions as a gate controlling access to the active site. The complex structure reveals a closed conformation, suggesting that substrate binding seals the active-site entrance so that the catalytic groups are sequestered from solvent. A comparison of the crystal structures of the bacterial and fungal cytosine deaminases provides an elegant example of convergent evolution, where starting from unrelated ancestral proteins, the same metal-assisted deamination is achieved through opposite chiral intermediates within distinctly different active sites.Cytosine deaminase (CD, 1 EC 3.5.4.1) catalyzes the deamination of cytosine to uracil and 5-methylcytosine to thymine. The enzyme has been found in bacteria and fungi, where it plays an important role in pyrimidine salvage. However, it is not present in mammalian cells, which utilize cytidine deaminase (CDA) instead (1). The bacterial and fungal CDs are distinct from each other and have evolved separately. The 426-residue hexameric Escherichia coli enzyme like the murine adenosine deaminase belongs to the (␤/␣) 8 -barrel amidohydrolase superfamily, in which four histidines and one aspartate located at similar spatial positions are conserved for metal coordination and enzyme catalysis (2-4). On the other hand, the 158-residue dimeric yeast counterpart may share two conserved signature sequences, HXE and CXXC, with a variety of deaminases, and thus has been grouped into the cytidine and deoxycytidylate deaminase family in the Pfam protein family data base (5, 6). The crystal structure of E. coli CDA reveals that the signature sequences contain a zinc binding motif, with histidine and two cysteines acting as zinc ligands while the glutamate serves as a proton shuttle (7).The antimetabolite 5-fluorouracil (5-FU) is one of the most active chemotherapeutic agents for the treatment of colorectal cancer, but it has limited efficacy due to gastrointestinal and hematological toxicities (8). Because of its ability to convert the relatively nontoxic 5-fluorocytosine (5-FC) into 5-FU and its absence in mammalian cells, CD has become an attractive candidate for the reduction of 5-FU toxicity toward n...

The structure of a yeast RNA-editing deaminase provides insight into the fold and function of activation-induced deaminase and APOBEC-1

“…3B). These data supported a model for CDA activity in which the active-site flap restricted substrate access to the active site as observed for ScCD (30). The observation that both the ScCDD1-EcL19-CDD1* and ScCDD1-AL-⌬(␣1)-CDD1* constructs deaminated cytidine in vitro demonstrated that the purified recombinant enzymes exhibited both folding and catalytic capabilities.…”

“…The tertiary fold of the CDD1 monomer (Fig. 1B) exhibited high structural homology to the catalytic domains of known bacterial CDAs from E. coli and B. subtilis (29), as well as ScCD (30), despite modest sequence identity; respective rms deviation values from superpositions were 1.32 Å (28% identity), 0.95 Å (43%), and 1.42 Å (16%). The tertiary fold of the deaminase catalytic domain was a triangular ␤-sheet comprising five core strands flanked on either broad face by three ␣-helices (Fig.…”

“…1D). In contrast, the C 2 subunit interface of dimeric Sc cytosine deaminase (CD) does not appear critical for enzymatic activity because each monomer possesses a self-contained active site (30). The simplicity of the base substrate may have allowed divergence in the ScCD domain that led to the acquisition of a large C-terminal flap (Fig.…”

“…Misalignment was likely because the N-terminal 48-aa helix bundle from EcCDA does not appear in the structures of either ScCDA, B. subtilis CDA, or ScCD (29,30), and therefore should not be considered part of the core deaminase fold. The need for new APOBEC-1-related protein models is underscored by the fact that the EcCDA-based model has been adopted by other laboratories attempting to elucidate the structure and function of APOBEC-1-related protein family members (36,41), but has been criticized subsequently for lack of structural rigor (21,35).…”

“…An alignment template was constructed by use of a quadruple C ␣ superposition of CDAs including: ScCDD1 (this study), E. coli (Ec)CDA (28), Bacillus subtilis (Bs) CDA (29), and the ScCD monomer (30). Pairwise matches of tetrameric ScCDD1 atoms with the tetrameric enzyme of B. subtilis (29) and the dimeric enzyme from E. coli (28) produced rms deviation values of 1.14 and 1.54 Å for 88% and 57% of spatially matched atoms within the CDA core.…”