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J. Biol. Chem., Vol. 281, Issue 11, 7605-7613, March 17, 2006

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Crystal Structure of a Bifunctional Deaminase and Reductase from Bacillus subtilis Involved in Riboflavin Biosynthesis^*

Sheng-Chia Chen¹, Yuan-Chih Chang¹, Chao-Hsiung Lin^¶, Chun-Hung Lin^||, and Shwu-Huey Liaw^¶**2

From the Structural Biology Program, Institute of Biochemistry, and ^||Faculty of Life Science, National Yang-Ming University, Taipei 11221, Taiwan, ^¶Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan, and ^**Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei 11217, Taiwan

Received for publication, September 19, 2005 , and in revised form, November 3, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Bacterial RibG is an attractive candidate for development of antimicrobial drugs because of its involvement in the riboflavin biosynthesis. The crystal structure of Bacillus subtilis RibG at 2.41-Šresolution displayed a tetrameric ring-like structure with an extensive interface of 2400 Ų/monomer. The N-terminal deaminase domain belongs to the cytidine deaminase superfamily. A structure-based sequence alignment of a variety of nucleotide deaminases reveals not only the unique signatures in each family member for gene annotation but also putative substrate-interacting residues for RNA-editing deaminases. The strong structural conservation between the C-terminal reductase domain and the pharmaceutically important dihydrofolate reductase suggests that the two reductases involved in the riboflavin and folate biosyntheses evolved from a single ancestral gene. Together with the binding of the essential cofactors, zinc ion and NADPH, the structural comparison assists substrate modeling into the active-site cavities allowing identification of specific substrate recognition. Finally, the present structure reveals that the deaminase and the reductase are separate functional domains and that domain fusion is crucial for the enzyme activities through formation of a stable tetrameric structure.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Flavin coenzymes are ubiquitous in all organisms because of their involvements in central metabolic pathways. Plants and many microorganisms obtain the precursor riboflavin by biosynthesis, whereas animals depend on nutritional sources. Numerous pathogenic microorganisms are unable to take up flavins from the environment and hence are absolutely dependent on their endogenous production. Therefore, the enzymes involved in riboflavin biosynthesis have the potential to become attractive candidates for the design of new defenses against antibiotic-resistant pathogens.

During riboflavin biosynthesis (1), GTP cyclohydrolase II first catalyzes the hydrolytic C-8 release of GTP to yield formate and pyrophosphate as side products. The product, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (compound 1) is converted into 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate (compound 4) by deamination of the pyrimidine ring and NAD(P)H-dependent reduction of the ribose (Fig. 1). The deamination and reduction steps have been shown to proceed in the opposite order in yeast and Escherichia coli (2, 3). Most eubacteria contain a bifunctional protein; for instance Bacillus subtilis RibG (BsRibG)³ is composed of an N-terminal deaminase domain (D domain) and a C-terminal reductase domain (R domain) (4). In contrast, in fungi, plants and most archaea, these two enzymes are separate (5-7). In yeast, Rib7 is the corresponding reductase, whereas the C-terminal domain of Rib2 is responsible for deamination, in addition to N-terminal pseudouridine synthase activity.

The D domain of bacterial RibG was expected to belong to the cytidine deaminase (CDA) superfamily because of the two conserved signatures, H(C)XE and PCX_8-9C. The CDA superfamily consists of the mononucleotide deaminases involved in nucleotide metabolism, and the RNA/DNA-editing deaminases involved in gene diversity and in antivirus defense (8, 9). The RNA/DNA-editing deaminases include A- to -I tRNA-specific adenosine deaminases (TADs), A- to -I adenosine deaminases acting on RNA, and C- to -U cytidine deaminases acting on RNA/DNA. These deaminases catalyze the hydrolytic deamination of cytosine, guanine, and adenine moieties and several of their therapeutically useful analogues. The available member structures reveal a virtually identical zinc-assisted deamination mechanism with the consensus histidine and cysteines acting as the zinc ligands, whereas the glutamate serves as a proton shuttle (10-14). However, the question of how nature has evolved the CDA fold into these various deaminases requires additional study.

Furthermore, the structural fold of the R domain was predicted by 3D-PSSM (15) to be similar to dihydrofolate reductase (DHFR). DHFR catalyzes the NADPH-utilizing reduction of dihydrofolate to tetrahydrofolate. Many DHFR inhibitors, such as methotrexate, pyrimethamine, and trimethoprim, have long been used clinically in the treatment of cancer, rheumatoid arthritis, malaria, and bacterial and fungal infection (16). Therefore, the R domain may become an important target for new drug design. To gain structural insights into the inhibitor design, substrate specificity, and evolution, we have solved the BsRibG structure at 2.41-Å resolution.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Protein Preparation—The His-tagged BsRibG was expressed using the pQE30 vector (Qiagen) in E. coli BL21 pLysS. The recombinant protein contains 12 additional vector residues (MRGSH₆GS) at the N terminus. One-liter Luria broth (LB) cultures were grown at 37 °C to an A₆₀₀ of 0.8 and induced with the addition of 1 mM isopropyl -D-thiogalactopyranoside. Cells were grown for another 5 h at 37°C before harvest. Cell pellets were resuspended in 40 ml of cold buffer A (20 mM Tris-HCl, 200 mM NaCl, pH 7.5) and lysed by French press. After removal of cellular debris by centrifugation at 39,000 x g at 4 °C for 30 min, the supernatant was applied to a 5-ml nickel-nitrilotriacetic acid column (Ni-NTA, Qiagen). The resin was washed with 40 mM imidazole in buffer A, and the protein was eluted with buffer A containing 500 mM imidazole. The protein fractions were collected, dialyzed against buffer B (20 mM Tris-HCl, pH 8.5), and loaded onto a Sepharose^TM Q column (Amersham Biosciences). After washing with 230 mM NaCl in buffer B, the BsRibG was eluted with 260 mM NaCl and dialyzed against 20 mM HEPES (pH 7.5) and 5 mM dithiothreitol.

View larger version (7K): [in this window] [in a new window]   FIGURE 1. The deamination and reduction steps in the riboflavin biosynthesis.

Protein Crystallization—The initial crystallization screening was performed with screening kits using the hanging-drop vapor diffusion method at 22 °C. The hanging drops were mixtures of 2 µl of reservoir solution and 2 µl of protein solution. Protein crystals could be obtained under several reservoir solutions, and the best crystals were grown in 26.6% polyethylene glycol 400, 190 mM MgCl₂, 5% glycerol, and 95 mM HEPES (pH 7.5) with a protein solution of 20-25 mg/ml. Crystals appeared and reached their final dimensions in 1 week at 15 °C. The NADPH derivative was prepared by soaking crystals for 4 days in reservoir solution containing 10 mM NADPH. X-ray diffraction data were collected and processed at beamlines BL12B2 at SPring-8 (Harima, Japan) and NW12 at the Photon Factory (Tsukuba, Japan). The crystals belong to the P2₁2₁2₁ space group with 1 tetramer/asymmetric unit.

Structure Determination—The BsRibG structure was determined using single-wavelength anomalous dispersion (SAD) of the endogenous zinc ion. Four Zn²⁺ positions were identified and refined with SOLVE (18). The initial phase was improved by direct-method phasing refinement using OASIS (19) before density modification. The initial electron density map showed an interpretable density of 70% of the tetramer. About 60% of the initial tetrameric model was automatically built using RESOLVE (18). Because the structural fold was predicted to be similar to yeast cytosine deaminase (yCD) and Thermotoga maritima DHFR (TmDHFR) (10, 20), both structures were used to assist in the manual building of the atomic model using TURBO-FRODO (21), and refinement was carried out against the native data to 2.41-Å resolution using crystallography NMR software (22). The statistics for data collection and refinement are summarized in Table 1. About 88% of the residues are in the most favored regions of the Ramachandran plot, with the remaining in the additional allowed regions.

View larger version (40K): [in this window] [in a new window]   FIGURE 2. Sedimentation velocity analysis of BsRibG. The enzyme concentration was 0.3 mg/ml in 20 mM HEPES (pH 7.5). The three panels represent the optical traces (top), the residues of the model fitting (middle), and the sedimentation coefficient distribution (bottom), respectively. The ultracentrifugation analysis demonstrated that BsRibG exists in solution as a tetramer with a protein concentration ranging from 80 to 300 µg/ml.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Overall Structure—Analytical ultracentrifugation experiments clearly demonstrated that BsRibG exists as a tetramer in solution as well as in crystal form, where the enzyme forms a tetrameric ring-like structure (Figs. 2 and 3a). The current tetrameric model contains residues 1-359 for each subunit. Each monomer is composed of two separate functional domains (Fig. 3b). The N-terminal D domain (residues 1-143) consists of a central five-stranded -sheet (1-5) with 1 running antiparallel to the others. The -sheet is sandwiched by two helices (A and E) on one side and by three helices (B, C, and D) on the other side. The C-terminal helix F extends away from the D domain and connects to the R domain. The R domain (residues 146-359) is composed of a large nine-stranded -sheet (A-H and D') with the C-terminal strand H running antiparallel to the others. The -sheet is flanked by five -helices (B, C, D', E, and F). The secondary structure elements of the R domain are numbered as for DHFRs (28).

The four subunits in the crystal asymmetric unit did not show significant differences between each individual domain except for several loops (root-mean-square deviations (r.m.s.d.) of 0.38-0.46 and 0.59-0.73 Šfor the backbones of residues 2-139 and residues 146-359, respectively). However, the relative orientations between the D and R domains are slightly different, resulting in a weak noncrystallographic symmetry. Molecules A and B interact with each other through their D domains with a buried surface area of 650 Ų per D domain (the D interface) (Fig. 4A), whereas molecule A makes extensive contacts with molecule C through their respective R domains with a buried area of 1750 Ų per R domain (the R interface) (Fig. 4B). There are no contacts between molecules A and D in the tetramer.

The D interface is made up mainly of the N-terminal two helices A and B, the 2 strand, and the connected loops (residues 4-19 and 35-61). There are 14 direct hydrogen bonds between the protein atoms across the interface and hydrophobic patches formed by Leu⁸, Leu¹², Ile³⁶, Met³⁹, Leu⁴³, and Met⁵⁷. The anti-parallel disposition of the two pseudodyad-related A helices also contributes to the interface by dipole-dipole interactions. Interestingly, the side chains of His⁵⁶ from the two D domains stack very well, with a distance of 3.3-3.4 Å between the aromatic rings. The R interface mainly is made up of: the two large loops, L_A-B (residues 156-169) and L_F-G (residues 314-338); and the C-terminal residues 339-358. There are 18 direct hydrogen bonds between the protein atoms across the interface, and extensive hydrophobic patches are by Try¹⁶⁹ formed, Pro³¹⁴, Lys³¹⁵, Leu³¹⁶, Ile³¹⁷, Leu³²⁵, Phe³³¹, Met³³⁴, Val³³⁷, Leu³³⁹, Leu³⁴⁰, Phe³⁴², Ile³⁴⁵, Ile³⁵², and Leu³⁵⁴.

View larger version (63K): [in this window] [in a new window]   FIGURE 3. Structure of BsRibG. Ribbon views of the tetramer (a) and monomer (b). The tightly bound zinc ion in D domain is shown as a sphere (magenta) with the cofactor NADPH (black) and the modeled substrates (cyan) as ball- and -stick representations. Molecules A-D in the tetramer, as referenced in the text, are colored in red, blue, green, and black, respectively. The D and R domains are made up of a central -sheet flanked by -helices. These two domains have few contacts with each other and hence can fold independently.

The active site of the D domain contains one tightly bound endogenous zinc ion, for which the anomalous data provided sufficient phase information for structure determination. The zinc ion is tetrahedrally coordinated by His⁴⁹ N¹ (2.0 Å), Cys⁷⁴ S (2.4 Å), Cys⁸³ S (2.3 Å), and a water molecule (2.0 Å).The zinc-bound water molecule interacts with Glu⁵¹ O² (2.5 Å). The active-site architecture resembles those of the CDA members, which share a similar zinc-assisted deamination mechanism with a virtually identical interaction network between the common moiety of the pyrimidine ring of the substrate, the zinc ion, the zinc-bound water molecule, the zinc ligands, and the base glutamate (Fig. 5B). In addition, the active-site cavities of these deaminases are mainly made up of the C-terminal tail and the loops connecting the A-1, 2-B, 3-C, and 4-D (Fig. 5C).

Based on the structural comparison of the active-site cavities, the substrate of the D domain was modeled into the active site through superposition of the nucleobase rings because of the highly conserved interaction networks surrounding the target amino group (Fig. 5B). The model was then subjected to energy minimization with crystallography NMR software. Simulation of the complex structure suggested that the nucleophilic OH-2 group of the pyrimidine ring coordinates to the catalytic zinc ion and interacts with Glu⁵¹O² and Cys⁷⁴ N. The NH-3 group hydrogen bonds with Glu⁵¹ O¹, the O-4 atom with Ala⁵⁰ N and His⁴² N, and the NH₂-5 with Asn²³ O¹ and His⁴² N¹. The two hydroxyl groups of the ribose have close contacts with the side chains of Asp¹⁰¹ and Asn¹⁰³. The phosphate moiety forms salt bridges with His⁷⁶ and Lys⁷⁹, located in the unique insertion between the two zinc ligand cysteines, and these interactions are essential for the deamination activity because the enzyme cannot utilize the dephosphorylated form as substrate (3). These predicted substrate-binding residues are all highly conserved in the eubacterial RibGs. However, the fungal deaminases such as yeast Rib2 (yRib2) apparently contain different substrate-interacting residues because of their distinct substrate, which has an open ribityl group instead of a cyclic ribose (Figs. 1 and 5C).

View larger version (51K): [in this window] [in a new window]   FIGURE 4. Subunit interfaces. Stereo views of the D interface (A) and the R interface (B). Two distinct subunit interfaces are formed by the D and R domains with total buried areas of 1300 and 3500 Å2, respectively. The D interface is made up mainly of the 2 strand and helices A and B, whereas the R interface is made up of the two large loops, LA-B and LF-G, and the C-terminal tail.

View larger version (57K): [in this window] [in a new window]   FIGURE 5. Structural conservation and divergence of the CDA superfamily. A, stereo view of structural superposition of BsRibG (red), yCD (blue) (10), T4dCMPD (green) (13), and AaTADA (yellow) (14). The zinc ion is displayed as a sphere (magenta) with the yCD inhibitor 3,4-dihydrouracil (DHU)(cyan) shown as ball- and -stick representations. These deaminases share the conserved -sheet and helices A-C and even a part of the E helix. T4dCMPD contains a 60-residue insertion, which folds into two helices (B' and B'') and flexibleloops. The C-terminal tail of yCD folds backward to limit the pocket size, whereas those of the remaining members swing away to enlarge the active-site cavity. B, superposition of the active sites of BsRibG (magenta), yCD (cyan), and T4dCMPD (green). The residue numbering is labeled in the same color for each protein. The deaminases display highly conserved interaction networks surrounding the target amino group of the nucleobase ring. In contrast, each member contains its own unique substrate recognition residues. C, multiple sequence alignment of some CDA members. The Arabidopsis thaliana deaminase (At363) (6) involved in riboflavin biosynthesis is also included. The GenBankTM accession codes are listed in the right column. Secondary structure elements for BsRibG are labeled (s.s). The number of residues in gaps is indicated in parentheses, and the protein length is in brackets. The superfamily signatures, H(C)XE and PCX2-9C, are shaded in cyan, whereas the residues for the conserved hydrophobic core are in yellow. In addition, the substrate-binding residues in the known complex structures are shaded in red, and those that are predicted are in blue. The unique signatures for each member are highlighted in italics, and the residues involved in loss-of-function point mutants of AID are shaded in magenta.

Structure-based Sequence Alignment of the CDA Members—In combination with a sequence-structure analysis, a structure-based sequence alignment of the CDA members was constructed (Fig. 5C). Multiple sequence alignments reveal that there are unique member signatures that are useful for gene annotation. The unique substrate recognition residues for the cytosine deaminases are WXXDI at the C terminus, and those for the guanosine deaminases are FDD between helices D₁ and D₂ and a tyrosine at the C terminus. The CDAs also have unique substrate-binding residues, namely FXV at the N terminus of the 1 strand and NXE at the C terminus of the 2 strand. The absence of the D helix between the 4 and 5 strands causes both strands to become antiparallel and the C terminus to be in the opposite direction. dCMPDs possess GYNG on the 2 strand and a diverse extra 20-60-residue insertion between the 2 strand and the B helix. Interestingly, most phosphate-interacting residues are located in this insertion and are not conserved across dCMPDs. RibGs have NPXVG at the N terminus of the 1 strand and an extra 8-9-residue loop between the two zinc-ligating cysteines (PCX_8-9C). The extra loop is highly conserved in RibGs and is predicted to interact with the phosphate moiety of the substrate.

Even though the CDA members display substantial sequence diversity (15-25% sequence identity), our structure-sequence analysis suggests that comparative modeling is feasible. For instance, bacterial TADAs and eukaryotic TAD2s share a unique signature, EXPVG, at the N terminus of the 1 strand, in which the glutamate is also conserved in TAD1. Our comparative modeling suggests that this glutamate residue may serve as an adenine recognition residue for these A- to -I tRNA deaminases.

View larger version (28K): [in this window] [in a new window]   FIGURE 6. The NADPH-binding site in the R domain. A, the 2Fo-Fc electron density map for NADPH contoured at 1.5 level and is shown in cyan. B, schematic diagram of BsRibG interactions with the NADPH cofactor. Hydrogen bonds are presented as dashed lines; the interatomic distances are given in angstroms. "Radiating" spheres indicate hydrophobic contacts between the cofactor and the surrounding residues.

View larger version (56K): [in this window] [in a new window]   FIGURE 7. Structural conservation and divergence between RibG and DHFR. A, stereo view of structural superposition of the R domain of BsRibG (red) and TmDHFR (green) (20). The R domain contains an extra 25-residue insertion, which folds into the D' helix and the D' strand. B, multiple sequence alignment. Three other reductases involved in the riboflavin biosynthesis from A. thaliana (At599), yeast (5), and the Archaea Methanocaldococcus jannaschii (7) and two DHFRs from hyperthermophilic T. maritima and mesophilic E. coli are also included. The SwissProt accession codes are listed in the right column. Residues involved in NADPH binding are shaded in magenta, whereas the residues for the conserved hydrophobic core are in yellow. The substrate-binding residues in the known complex structures are shaded in red, and those that are predicted are in blue. DHFRs and RibG share four conserved regions for NADPH binding (motifs A-D). Their residues involved in substrate recognition are located in similar spatial positions in the A strand, the B helix, and the loop between the C helix and the C strand.

NADPH Binding in the R Domain—NADPH was identified by its strong electron density and is firmly embedded at the active site of the R domain (Fig. 6A). The enzyme did not show significant structural changes upon NADPH binding, with an r.m.s.d. of 0.4 Å for all of the backbone atoms. The cofactor is bound in an extended conformation with extensive interactions with the protein. The adenine N-3 and N-6 atoms interact with Thr²²¹ and Ser²⁹⁸, respectively (Fig. 6B). The phosphate group at the ribose O-3' makes close contacts with Thr²²¹. These interactions seem not to contribute significantly to the cofactor binding, because NADH as well as NADPH can serve as the coenzyme (4). The pyrophosphate moiety interacts with a constellation of backbone NH groups from Gly¹⁹⁴, Thr¹⁹⁵, Gly²⁹², Ser²⁹³, Ala²⁹⁴, and Val²⁹⁵ and the side chain of Thr¹⁹⁵. These residues are located at the N termini of helices C and E, and the helix dipoles may partially neutralize the negative charges of the phosphate groups. The nicotiamide amide group complementarily interacts with the amide backbone of Ala¹⁵³, whereas the nicotinamide ribose OH-2' and OH-3' contact with Asp¹⁹⁹ and Gly¹⁶⁵, respectively.

Structural Conservation and Divergence between BsRibG and DHFRs—A structural homology search by DALI (34) revealed that the R domain displays significant structural similarity to DHFRs (Fig. 7A). Despite low sequence identity, which is as little as 20%, all the DHFR structures exhibit a virtually identical fold consisting of a central eight-stranded -sheet (designated A-H) and four flanking helices (B, C, E, and F) (28). The R domain of BsRibG contains an extra 25-residue insertion between the D strand and the E helix, forming the D' helix and the D' strand. This insertion is quite a distance away from the active site and is diverse across the bacterial and fungal reductases.

TmDHFR is the most stable DHFR isolated thus far. The most prominent feature of TmDHFR is its highly stable dimer, which has been shown to make a major contribution to the high intrinsic stability (20). The R-R structure of BsRibG is similar to the dimeric TmDHFR structure. Both TmDHFR and BsRibG utilize similar regions for the subunit association. The contact areas of the R interface are larger than those of TmDHFR, 3500 Ų versus 3050 Ų, perhaps due to a larger L_F-G loop in BsRibG. Structural superposition of RibG and TmDHFR reveals an r.m.s.d. of 1.5 Šfor 120 C atoms with 25% sequence identity. The strong conservation of the tertiary structures suggests that the two reductases involved in the riboflavin and folate biosyntheses are descended from a single ancestral gene and there by define a new superfamily.

DHFRs and BsRibG share four conserved regions for NADPH binding, motifs A-D (Fig. 7B). In motif A, the two hydrogen bonds between the amide groups of the nicotinamide ring and an alanine residue at the C terminus of the A strand (Ala¹⁵³ in BsRibG) are strictly conserved. Both proteins use motifs B and D for the pyrophosphate binding, and motif C for the phosphate group at the adenosine O-3'. The interactions between DHFRs and the 3'-phosphate group are so extensive via 3-4 residues that the enzyme has a strict NADPH dependence. Both DHFR and RibG utilize many main chain atoms for NADPH binding, and hence the corresponding sequences of the NADPH-interacting residues have diverged during evolution, but their spatial positions have remained convergent in the four regions. A conserved cis peptide bond occurs at the two consecutive glycine residues in motif D. In contrast, both enzymes form diverse interactions with the remaining areas including the adenine ring and the riboses.

The enzymatic mechanism of DHFR has been chemically and structurally studied in detail (28, 35). A complete catalytic scheme through five kinetically observable intermediates has been proposed. Similarly, RibG is expected to catalyze the reduction of a cyclic ribosyl into an open ribityl group by hydride transfer from the C-4 atom of the nicotinamide ring of NAD(P)H to the C-1' of the ribose with concomitant protonation of O-5'. The binding architecture of the nicotinamide ring is virtually identical in DHFR and RibG. Thus, based on the structural comparison, the substrate of the R domain was modeled into the active site with subsequent energy minimization. Simulation of the complex structure suggested that the O-2 atom of the pyrimidine ring interacts with Lys¹⁵¹ N and the NH-3 and O-4 with Thr¹⁷² O¹. Thr¹⁹⁵ and Asp¹⁹⁹ make close contacts with the two hydroxyl groups of the ribose. Arg¹⁷⁶, Arg¹⁸³, and Arg²⁰⁶ form salt bridges with the phosphate group, interactions that are essential for the reduction activity because the enzyme cannot utilize the dephosphorylated form as substrate (3). These substrate-binding residues are highly conserved in the reductases and are located in spatial positions similar to those in DHFRs (Fig. 7B). Lys¹⁵¹ is highly conserved in most eubacteria and plants but not in fungi and archaea, implying that this residue distinguishes the O-2 atom from the NH₂-2 group of the pyrimidine moiety, which is the only difference in the substrates (Fig. 1). A highly conserved glutamate in motif D (Glu²⁹⁰ in BsRibG) may assist in proton transfer during the hydride transfer.

Fusion of Two Enzyme Domains in RibG—There is no evidence for any dependence of the active sites within the RibG tetramer. The active sites of the D and R domains within the same molecule are 40 Å apart if measured from the zinc ion to the nicotiamide C-4 atom. The openings of the D and R active sites point in the opposite directions. Unlike the bifunctional enzyme of DHFR and thymidylate synthase (36), the present BsRibG structure does not show a channel for substrate transport from the D domain to the R domain for the sequential reaction. In addition, the active sites of the dimeric TmDHFR have been shown to be independent (20). Unlike the monomeric DHFRs, TmDHFR does not show significant structural changes on binding of NADPH and the inhibitor methotrexate, because the active-site loops (L_A-B and L_F-G) in the monomeric enzymes are rearranged on the dimeric interfaces. Similarly, NADPH binding does not induce any significant conformational changes in BsRibG, and the corresponding loops are also involved in the R interface.

Previous deletion mutants have demonstrated that the N-terminal 147 residues and the C-terminal 248 residues of BsRibG were sufficient for their respective enzyme activities (4). However, these truncated proteins could not be isolated because of poor stability. The present structure reveals that the D and R domains make only a few contacts, including four hydrogen bonds by Glu²¹, Gln¹³⁵, His¹³⁹ Gln¹⁸⁷, Lys²¹², Gln³⁰⁶ and Lys³⁵⁷ and small hydrophobic contacts by Lys¹³⁶, Phe¹⁴⁰, Tyr¹⁴⁷, and Met²⁸⁵.If the F helix (residues 135-143) is considered as an interdomain linker, the D and R domain is even less interactive. Therefore, even though the two enzyme domains can fold independently, the domain fusion is crucial for the enzyme activities through formation of a stable tetrameric structure.

	FOOTNOTES

 
The atomic coordinates and structure factors (codes 2B3Z and 2D5N) 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 National Science Council Grant NSC94-2311-B010-017. 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.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Faculty 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.

³ The abbreviations used are: BsRibG, B. subtilis RibG; BsGD, B. subtilis guanine deaminase; CDA, cytidine deaminase; yCD, yeast cytosine deaminase; T4dCMPD, T4 bacteriophage dCMP deaminase; TAD, tRNA-specific adenosine deaminase; AaTADA, A. aeolicus tRNA-specific adenosine deaminase; DHFR, dihydrofolate reductase; TmDHFR, T. maritima dihydrofolate reductase; AID, activation-induced deaminase; APOBEC, apolipoprotein B mRNA-editing catalytic subunit; r.m.s.d., root-mean-square deviation; AICAR, aminoimidazole-4-carboxamide ribonucleotide.

	ACKNOWLEDGMENTS

 
We thank Dr. R. Kirby for critical reading of the manuscript and Dr. H. Chang for performing the ultracentrifuge experiments.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 

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