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J. Biol. Chem., Vol. 278, Issue 21, 19111-19117, May 23, 2003
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Crystal Structure of Yeast Cytosine Deaminase
INSIGHTS INTO ENZYME MECHANISM AND EVOLUTION*
¶
From the
Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan,
¶ Institute of Biopharmaceutical Science, National Yang-Ming University, Taipei 11221, Taiwan,
|| Institute of Biochemistry, National Yang-Ming University, Taipei 11221, Taiwan,
** Department of Life Science, National Yang-Ming University, Taipei 11221, Taiwan,
Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei 11217, Taiwan
Received for publication, January 27, 2003
, and in revised form, March 11, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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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 Å2 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 His62, Cys91, and Cys94 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.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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Cytosine deaminase (CD,1 EC 3.5.4.1 [EC] ) 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, 3, 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 normal distal tissues in enzyme-prodrug gene therapy (9). The combination of 5-FC with CD has been shown to effectively control tumor growth in animals and is currently being evaluated in several human clinical trials (10, 11). Previous studies also demonstrate that the use of yeast CD instead of the E. coli enzyme significantly improves treatment efficacy, perhaps due to the higher conversion efficiency of the 5-FC into 5-FU by the yeast CD (12, 13). Yeast CD, therefore, appears to be a better candidate for gene therapy. To provide insights into the catalytic mechanism, evolution, and gene therapy applications of the yeast CD, we have refined the enzyme structure to 1.6-Å resolution.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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The protein crystallization, diffraction data collection, phase determination, and building of an initial dimeric model with 218 alanine residues have been described in our preliminary report (14). However, subsequent inclusion of additional 52 residues and most side chains as well as the inhibitor and zinc ion into the model did not yield satisfactory results upon further refinement. Subsequent modification, analysis, and computational refinement of the model including simulated annealing were carried out using O (15) and CNS (16). Five percent of randomly selected reflections were set aside for calculating Rfree to monitor the progress of the refinement. An R-value of 0.45 at 1.6 Å resolution was obtained after rigid-body refinement of the model, and a difference Fourier map of 2Fo - Fc was computed. In most regions of this map, the polypeptide backbones and side chains can be clearly seen. The protein model was modified accordingly. However, in both subunits the region corresponding to amino acid residues 48–62 showed a degree of disordered peptide modeling that did not match the electron densities. Non-crystallographic symmetry averaging and density modification of the map did not make much improvement. In the end a model was reconstructed manually with alternating reference to the densities of both subunits. The model was then refined with strong non-crystallographic symmetry restraints, yielding R and Rfree values of 0.285 and 0.317, respectively. The resulting electron density map was much better. After the addition of water molecules and extension of the terminal polypeptides in the model, the refinement proceeded with release of the non-crystallographic symmetry restraints.
Statistics for the refined model are shown in Table I. More than 92% of the residues are in the most favored regions of the Ramachandran plot, with the remaining in the additional allowed regions. An anomalous data set at the absorption edge of zinc was collected to 1.9-Å resolution for completeness and Rmerge values of 98.8 and 5.3%. The anomalous difference Fourier map for zinc was calculated using phase angles (-90°) from the "native" model. With almost identical unit cell dimensions, the initial R was 0.183, and it decreased immediately to 0.178 after rigid-body refinement at 1.9 Å. Two unique peaks of more than 50 
levels were seen in the map, which correspond to locations of the zinc ions. Figs. 1, 3, B and C, and 4A were generated by MolScript (17) and Raster3D (18), Fig. 2B was generated by Insight II (Molecular Simulations Inc.), Fig. 3A was generated by BobScript (19), and Fig. 4, B and C was generated by Grasp (20).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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The Overall Structure—The current model contains residues 8–158 with clear electron density. The protein structure is composed of a central five-stranded
-sheet (1-5) with the strand order 2, 1, 3, 4, 5 and with 1 running antiparallel to other strands. The -sheet is sandwiched by two -helices (A and E) on one side and by three -helices (B-D) on the other side (Fig. 1A). The enzyme forms a tightly packed dimer in the crystallographic asymmetric unit (Fig. 1B), without significant differences between the two subunits (root mean square deviations of 0.67 Å for all protein atoms). The dimer interface, constituted mainly by the helical layer (B-D) and the C-terminal tail, is extensive and buries 1450 Å2 of the 7450-Å2 molecular surface area per monomer. At least 26 residues from each subunit are involved in dimer formation. There are 18 direct hydrogen bonds between the protein atoms across the dimer interface, including four salt bridges between Arg73 and Glu154 and Asp92 and Arg125.
In the dimer center, two small hydrophobic patches are formed by Met93, Ala97, and Met100 and a buried water molecule is surrounded by Thr60, Ile65, and Met93. The anti-parallel disposition of the two dyad-related D helices also contributes to the interface by dipole-dipole interactions. Interestingly, the side chains of Tyr121 from the two subunits stack very well, with a distance of 3.6 Å between the aromatic rings, and enclose four water molecules in a cavity that has a 62-Å3 volume between helices C and D and is flanked by the two salt bridges between Asp92 and Arg125.
A New Structural Superfamily—A structural homology search by DALI (21) reveals that despite the apparent lack of sequence similarity, yeast CD displays significant structural similarity to chicken ATIC (22) and to Bacillus subtilis and E. coli CDAs (7, 23), with Z-scores of 11.3, 9.0, and 8.2, respectively. ATIC is a 64-kDa bienzyme, responsible for the final steps of de novo purine biosynthesis. It contains an N-terminal inosine 5'-monophosphate cyclohydrolase domain and a C-terminal 5-amino-4-imidazolecarboxamide ribonucleotide (AICAR) transformylase domain. The AICAR transformylase domain is made up of subdomains 2 and 4, with a large insertion (residues 469–532, subdomain 3) in subdomain 4. The insertion does not appear to be directly involved in the enzyme activity (24), being only present in the eukaryotic ATICs but not in the prokaryotic enzymes. On the other hand, two types of CDAs have been identified; they are a dimeric form such as E. coli CDA that contains a catalytic domain with a non-catalytic C-terminal domain and a tetrameric form such as B. subtilis CDA that only has a catalytic domain. The dimeric CDA may have arisen by a gene duplication of tetrameric CDA because there is pronounced structural similarity between the catalytic and the C-terminal domains (7, 23).
Detailed structural comparisons reveal that yeast CD displays a similar fold to those involving the following five segments: subdomain 2 of the AICAR transformylase domain, subdomain 4 of the AICAR transformylase domain, the catalytic domain of E. coli CDA, the C-terminal domain of E. coli CDA, and the single catalytic domain of B. subtilis CDA. The main-chain atoms of the 60–62 structurally equivalent residues overlay with root mean square deviations of 1.11, 1.72, 1.34, 1.21, and 1.00 Å and with 7, 5, 17, 15, and 22% sequence identity, respectively (Fig. 2). Surprisingly, even though yeast CD shares a similar active site and a high sequence identity with the CDAs, its closest structural match is the subdomain 2 of the AICAR transformylase domain. This is perhaps due to the common D helix and the same direction of 5 strand. Yeast CD and subdomains 2 and 4 of the AICAR transformylase domain contain common four helices A-D, whereas the CDAs only have three helices, A-C. Only the strand 1in yeast CD and AICAR transformylase run antiparallel to the others, whereas both 1 and 5 are antiparallel in CDAs. There are only four -stands (1-4) in the C-terminal domain of E. coli CDA.
The strong conservation of the tertiary structures of these domains suggests that they are descended from a single ancestral gene and thereby define a new superfamily. This gene may have been duplicated and fused to form E. coli CDA and the AICAR transformylase domain, with subsequent evolution causing significant divergence. For example, yeast CD and CDAs have developed a zinc binding motif for their metal-assisted catalysis. The orientations between helices B and C in the C-terminal domain of E. coli CDA and in the subdomains 2 and 4 in the AICAR transformylase domain are different from those of yeast CD and CDAs, perhaps due to the lack of a zinc binding motif. The missing D helix in CDAs has perhaps caused the 5 strand to evolve to be antiparallel to 4 and the C terminus to be in an opposite direction (Fig. 2B). The C-terminal copy in E. coli CDA retains its structural integrity but without any catalytic functionality. On the other hand, subdomains 2 and 4 of the AICAR transformylase have developed another enzyme mechanism (24).
The Active-site Architecture and Substrate Binding—The active site contains one tightly bound metal ion even though no metal ions were added either during protein purification or crystallization. The metal ion was identified by a very strong spherical electron density in the Fourier map and was assigned as zinc based on the zinc anomalous data (Fig. 3A). The zinc ion is tetrahedrally coordinated by His62 N
1 (2.06 Å), Cys91 S
(2.31 Å), Cys94 S (2.25 Å), and the hydroxyl group of the bound inhibitor (2.08 Å) (Fig. 3B). The protein ligands are located on the N termini of the B and C helices, and the helix dipoles may partially neutralize the negative charges of the cysteinate ligands to lower the pKa value of the nucleophilic water molecule (23).
2-Hydroxypyrimidine has been shown to be converted by E. coli CD into 4-(S)-hydroxyl-3,4-dihydropyrimidine (or 3,4-dihydrouracil), a hydrated adduct, and this acts as a transition-state intermediate analog (2). Similar inhibitors have been used in previous crystallographic studies of murine adenosine deaminase (3, 25) and E. coli CDA (7, 26). In the structure presented here, the inhibitor 2-hydroxypyrimidine is firmly embedded in the active site as the hydrated form and is identified by its strong electron density that correlates well with the shape of a pyrimidine ring. The orientation of the pyrimidine ring is unambiguous because of clear electron density bulges for the 2-keto oxygen (O2) and the hydroxyl group at position 4 (OH4) (Fig. 3A) and because of its extensive hydrogen bond interactions and hydrophobic contacts (Fig. 3B).
The OH4 group of the pyrimidine ring, which is an analog to the nucleophilic hydroxyl in the reaction transition state, directly coordinates to the catalytic zinc ion and interacts with Glu64 O
2 (2.45 Å) and Cys91 N (3.10 Å). Additionally, the N3 atom interacts with Glu64 O1 (2.81 Å), the O2 atom forms hydrogen bonds with Gly63 N (2.79 Å) and Asn51 N2 (2.95 Å), and the N1 atom contacts with Asp155 O1 (2.70 Å). In addition to the extensive hydrogen bond network described above, the imidazole ring of His62 stacks on the pyrimidine ring with an interplanar distance of 3.3 Å. Also, the C2, C5, and C6 atoms of the pyrimidine ring make close contacts with Ile33 C1 (3.20 Å), Lue88 C2 (4.36 Å), Phe114 C2 (4.08 Å), Trp152 Ch2 (3.74 Å), and Ile156 C2 (3.98 Å). Therefore, the structure of the complex presented here reveals that yeast CD converts the inhibitor 2-hydroxypyrimidine into 4-(R)-hydroxyl-3,4-dihydropyrimidine, which is enantiomeric to the configuration observed in E. coli CD; this will be discussed later (Fig. 3C). A Comparison of Yeast CD and CDAs—The active-site architectures of yeast CD and E. coli and B. subtilis CDAs share striking similarity (Fig. 4A). Superposition of their active sites reveals a very similar interaction network between the attacking water molecule, the zinc ion, the zinc ligands, and the proton shuttle. Thus, the four highly conserved residues in the signatures, HXE (or CXE), and CXXC, of the cytidine and deoxycytidylate deaminase family (5, 6), are responsible for generation of the attacking hydroxide ion and for enhancement of the attacked substrate susceptibility during the enzyme catalysis, suggesting a similar zinc-assisted deamination mechanism in the family.
On the other hand, each member in the family should contain its unique substrate recognition residues, for example, the C-terminal tail in yeast CD. Cytosine has been shown to be a slow substrate for E. coli CDA, whereas cytidine is not a substrate for yeast CD (27, 28). The position of the pyrimidine ring of the inhibitor in yeast CD is structurally equivalent to that in the CDAs (Fig. 4). However, several interactions between the inhibitor and enzyme in yeast CD are different from those in the CDAs. The conserved asparagine forms a hydrogen bond with the OH3' of the ribose in the CDAs, whereas it interacts with the pyrimidine 2-keto oxygen in the yeast CD. The imidazole ring of the conserved histidine for zinc ligation, His102 in the E. coli CDA, does not stack on the inhibitor pyrimidine ring. The ribosyl sugar of the cytidine cannot be accommodated within the active site of the yeast CD, because Asp155 occupies part of the ribose-binding site (Fig. 4).
Another major difference between yeast CD and the CDAs is a change in the mechanism of the active-site entrance. The structure reported here, the yeast CD, appears to have a closed conformation, because the C-terminal tail forms a "flap" across the entrance and, thus, caps the narrow opening of the activesite cavity (Fig. 4B). This flap may function as a gate that controls access to the active site and, thus, regulates both substrate binding and product release. The closed conformation described here may be due to direct interactions between the inhibitor and the Trp152, Asp155, and Ile156 residues of the C terminus, allowing sealing of the entrance. Unlike the active site of yeast CD, which is complete within the monomer itself, the active site in one monomer of the tetrameric B. subtilis CDA is made up of three subunits, and its entrance appears to be controlled by the C-terminal helix from the adjacent subunit (Fig. 4C). However, the flexibility of the C-terminal tail in the presence of the inhibitor is high, perhaps due to a lack of significant interaction with the inhibitor. This suggests that the C-terminal tail can easily swing out and allow substrate entry or product release (23). On the other hand, the active-site entrance switch in the dimeric E. coli CDA is more complicated and still unclear. In a single monomer complex, there are solvent-accessible areas of the inhibitor, but these are covered by three loops from the second monomer in the dimer, which complete the active site in the first monomer and form a lid on the active site (7).
A Proposed Catalytic Mechanism—On the basis of the structural studies on yeast CD and CDAs (7, 26, 29, 30), we propose a deamination mechanism for yeast CD as outlined in Scheme 1. First, the zinc ion activates the attacking water molecule to form a hydroxide ion with the assistance of Glu64. The substrate binds to the active site and induces a closed conformation to sequester the reaction from solvent. Glu64 serves as a proton shuttle, abstracting a proton from the zinc-bound water on one hand and subsequently protonating the N3 of cytosine on the other hand, thereby reducing the N3 to C4 double bond character and making the C4 atom more susceptible to the hydroxide ion for formation of the tetrahedral intermediate. Cleavage of the carbon-nitrogen bond is assisted by transferring the proton from OH4 to NH24 via Glu64 and a pyrimidinolate (O-) intermediate. The newly formed uracil may move toward the zinc ion for ligation, and this would weaken its interaction with the C-terminal tail, allowing its release from the active site.
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An Example for Convergent Evolution—The crystal structures of bacterial and fungal CDs provide an elegant example of convergent evolution in that they have evolved from unrelated ancestral proteins but have achieved the same deamination reaction (Fig. 3C). The two enzymes have completely different tertiary structures and distinct active sites, and they even catalyze the reaction through opposite chiral intermediates. However, both enzymes utilize a divalent cation to activate the water molecule and protein carboxylate group(s) for proton abstraction and donation. In yeast CD, the zinc ion is tetrahedrally coordinated, and there is only one carboxylate group (Glu64) to facilitate proton shuttling during catalysis. On the other hand, in E. coli CD, the iron ion is penta-coordinated, and the proton transfer is carried out by at least two different residues, Asp313 for proton abstraction from the nucleophile water and Glu217 for proton donation to the N3 atom (2). The different interaction networks between the two enzymes and the substrates lead to opposite enantiomeric configurations of their transition-state intermediates.
Previous studies demonstrate that yeast CD has a greater therapeutic potential as part of an enzyme-prodrug strategy in cancer patients compared with the bacterial enzymes. This is perhaps due to its more efficient conversion of 5-FC into 5-FU (12, 13). The Km value of E. coli CD for 5-FC is 8-fold higher than that for the natural substrate cytosine, whereas the Km of yeast CD for 5-FC is only one-fifth of that for cytosine (12). The structure presented here shows that the C5 atom of the pyrimidine ring is adjacent to the hydrophobic cluster, which includes Phe114, Trp152, and Ile156 (Fig. 3B). In particular, the C5 atom faces toward Phe114, with a shortest distance of 4.1 Å. A fluorine atom is attached to an aromatic ring will make it very hydrophobic. Therefore, the hydrophobic cluster in yeast CD should enhance the binding of 5-FC. On the other hand, the C5 atom faces toward Asp314 in E. coli CD, with a distance between C5 and Asp314 O1 of 3.6 Å. Close contact with such a polar residue would be unfavorable to 5-FC binding. These structural observations are consistent with the enzyme kinetic measurements.
Conclusion—In summary, the yeast CD structure shares high similarity to the AICAR transformylase domain, which may define a new superfamily. The enzyme structure also reveals a common zinc-assisted deamination for the cytidine and deoxycytidylate deaminase family. The unique C-terminal tail in yeast CD is involved in dimerization, substrate specificity, and as an active-site entrance switch. On the basis of the structural information, some protein engineering trials to obtain new functions through site-directed mutation and deletion of the C-terminal tail have been initiated.
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FOOTNOTES |
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The atomic coordinates and structure factors (code 1UAQ [PDB] ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
* This study was supported by Program for Promoting Academic Excellence of Universities Grant 89-B-FA22-2-4 (to J.-J. L.), an Academia Sinica Main Theme research grant (to A. H.-J. W.), Taipei Veteran General Hospital Grant VGH 91-376-6, and National Science Council Grant NSC 91-3112-B-010-012 (to S.-H. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
These authors contributed equally to the work.
To whom correspondence should be addressed: Dept. of Life Science, National Yang-Ming University, Taipei, Taiwan 11221. Tel.: 886-2-2826-7278; Fax: 886-2-2820-2449; E-mail: shliaw{at}ym.edu.tw.
1 The abbreviations used are: CD, cytosine deaminase; 5-FC, 5-fluorocytosine; 5-FU, 5-fluorouracil; CDA, cytidine deaminase; AICAR transformylase, 5-aminoimidazole-4-carboxamide ribonucleotide transformylase.
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ACKNOWLEDGMENTS |
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The synchrotron radiation experiments were performed at the National Synchrotron Radiation Research Center, Hsinchu, Taiwan, at the Photon Factory, Tsukuba, Japan, and at the SPring-8, Sayo, Japan. Thanks to Professor Ralph Kirby for help in preparing this manuscript.
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M. Kuratani, R. Ishii, Y. Bessho, R. Fukunaga, T. Sengoku, M. Shirouzu, S.-i. Sekine, and S. Yokoyama Crystal Structure of tRNA Adenosine Deaminase (TadA) from Aquifex aeolicus J. Biol. Chem., April 22, 2005; 280(16): 16002 - 16008. [Abstract] [Full Text] [PDF]
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 S. G. Conticello, C. J. F. Thomas, S. K. Petersen-Mahrt, and M. S. Neuberger Evolution of the AID/APOBEC Family of Polynucleotide (Deoxy)cytidine Deaminases Mol. Biol. Evol., February 1, 2005; 22(2): 367 - 377. [Abstract] [Full Text] [PDF]
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E. Johansson, M. Fano, J. H. Bynck, J. Neuhard, S. Larsen, B. W. Sigurskjold, U. Christensen, and M. Willemoes Structures of dCTP Deaminase from Escherichia coli with Bound Substrate and Product: REACTION MECHANISM AND DETERMINANTS OF MONO- AND BIFUNCTIONALITY FOR A FAMILY OF ENZYMES J. Biol. Chem., January 28, 2005; 280(4): 3051 - 3059. [Abstract] [Full Text] [PDF]
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S.-H. Liaw, Y.-J. Chang, C.-T. Lai, H.-C. Chang, and G.-G. Chang Crystal Structure of Bacillus subtilis Guanine Deaminase: THE FIRST DOMAIN-SWAPPED STRUCTURE IN THE CYTIDINE DEAMINASE SUPERFAMILY J. Biol. Chem., August 20, 2004; 279(34): 35479 - 35485. [Abstract] [Full Text] [PDF]
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 K. Xie, M. P. Sowden, G. S. C. Dance, A. T. Torelli, H. C. Smith, and J. E. Wedekind The structure of a yeast RNA-editing deaminase provides insight into the fold and function of activation-induced deaminase and APOBEC-1 PNAS, May 25, 2004; 101(21): 8114 - 8119. [Abstract] [Full Text] [PDF]
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