Human α-L-iduronidase uses its own N-glycan asa substrate-binding and catalytic moduleNobuo Maitaa,b,1, Takahiro Tsukimurac, Takako Taniguchib, Seiji Saitod, Kazuki Ohnoe, Hisaaki Taniguchib,and Hitoshi Sakurabaf,g

aLaboratory of X-Ray Crystallography, Institute for Enzyme Research, University of Tokushima, Tokushima 770-8503, Japan; bDivision of Disease Proteomics,Institute for Enzyme Research, University of Tokushima, Tokushima 770-8503, Japan; cDepartment of Functional Bioanalysis, Meiji Pharmaceutical University,Tokyo 204-8588, Japan; dDepartment of Medical Management and Informatics, Hokkaido Information University, Hokkaido 069-8585, Japan; eResearchSection, NPO for the Promotion of Research on Intellectual Property Tokyo, Tokyo 100-0005, Japan; fDepartment of Analytical Biochemistry, MeijiPharmaceutical University, Tokyo 204-8588, Japan; and gDepartment of Clinical Genetics, Meiji Pharmaceutical University, Tokyo 204-8588, Japan

Edited by Elizabeth F. Neufeld, David Geffen School of Medicine at University of California, Los Angeles, CA, and approved July 25, 2013 (received for reviewApril 12, 2013)

N-glycosylation is a major posttranslational modification thatendows proteins with various functions. It is established thatN-glycans are essential for the correct folding and stability of someenzymes; however, the actual effects of N-glycans on their activ-ities are poorly understood. Here, we show that human α-L-idur-onidase (hIDUA), of which a dysfunction causes accumulation ofdermatan/heparan sulfate leading to mucopolysaccharidosis type I,uses its own N-glycan as a substrate binding and catalytic module.Structural analysis revealed that the mannose residue of the N-gly-can attached to N372 constituted a part of the substrate-bindingpocket and interacted directly with a substrate. A deglycosylationstudy showed that enzyme activity was highly correlated with theN-glycan attached to N372. The kinetics of native and deglycosy-lated hIDUA suggested that the N-glycan is also involved in catalyticprocesses. Our study demonstrates a previously unrecognized func-tion of N-glycans.

X-ray crystallography | N-linked glycan | glycoside hydrolase family 39

Asparagine-linked protein glycosylation, one of the majorposttranslational modifications in eukaryotes, causes linking

of an oligosaccharide chain to the Nδ atom of an asparagine inthe N-glycosylation signal (Asn-Xaa-Ser/Thr, Xaa can be anyamino acid except proline). N-glycosylation endows proteins withseveral abilities including immune recognition, ligand-receptorbinding, and cell signaling, trafficking, folding, and stability (1–3). For some lysosomal enzymes such as glucocerebrosidase (4)and α-galactosidase A (5), the deglycosylation reduces the enzymes’activities, presumably through a reduction in protein stability.However, the precise relationships between N-glycans and en-zyme activities remain unknown (2, 6).α-L-Iduronidase (IDUA; EC 3.2.1.76) is a lysosomal enzyme,

and deficient activity of IDUA causes accumulation of glyco-saminoglycans in lysosomes leading to mucopolysaccharidosistype I (MPS I) (7). Human IDUA is translated as 653 aminoacids and N-glycosylated at six potential sites (N110, N190,N336, N372, N415, and N451), and then its N-terminal 26 resi-dues are removed and it is processed to the mature form inlysosomes (8, 9). IDUA hydrolyses the glycosidic bond betweenthe terminal L-iduronic acid (IdoA) and the second sugar of N-acetylgalactosamine (GalNAc)-4-sulfate/N-sulfo-D-glucosamine(GlcNS)-6-sulfate, which are the major components of dermatan/heparan sulfate (Fig. 1A). Thus, a defect of IDUA leads to excessstorage of dermatan/heparan sulfate and causes a systemic dis-order, MPS I, involving progressive mental retardation, grossfacial features, an enlarged and deformed skull, a small stature,corneal opacities, hepatosplenomegaly, valvular heart defects,thick skin, joint contractures, and hernias (10). The prevalence ofMPS I in England and Wales from 1981 to 2003 was 1.07 casesper 100,000 births (11). A recombinant human IDUA (hIDUA)expressed in Chinese hamster ovary (CHO) cells (marketed as

Aldurazyme) was developed for enzyme replacement therapy(12), which is widely used for MPS I treatment.IDUA belongs to glycoside hydrolase (GH) family 39 in the

CAZy database (13). To date, crystal structures of the bacterialGH39 β-xylosidase (XynB) have been reported (14, 15). In ad-dition, a homology model of hIDUA constructed from Ther-moanaerobacterium saccharolyticum XynB (PDB ID code 1PX8)has been reported (16). As the sequence homology betweenhIDUA and T. saccharolyticum XynB is quite low (28.4% simi-larity), the reliability of the model is not high. Recently, thecrystal structures of apo-hIDUA, expressed in a plant seed, weresolved. Nevertheless, the structure of hIDUA expressed inmammalian cells is strongly required for an insight into the basisof MPS I and the development of new therapies. In this study, weexplored the functions of the N-glycans in hIDUA. We foundthat the deglycosylation of hIDUA with endoglycosidase H(Endo H), but not peptide-N-glycosidase F (PNGase F), reducesthe enzyme’s activity. Concanavalin A (ConA) pull-down assaysuggested that PNGase F–resistant N-glycans are essential forthe enzyme activity of hIDUA. We also solved the crystalstructures of hIDUA alone and in a complex with IdoA: theyrevealed that the N-glycan attached at N372 makes up one sideof the substrate-binding pocket and directly interacts with theIdoA. Further, we found that the enzyme activity showed highcorrelation with the amount of N-glycan at N372. The kineticsof native and deglycosylated hIDUA implied that the N-glycanis also involved in the catalytic process. Our finding indi-cates a previously unrecognized function of N-glycans in theenzyme activity.

ResultsActivity of hIDUA Is Reduced on Endo H but Not PNGase F Treatment.To examine the influence of N-glycans on hIDUA activity, wecarried out a deglycosylation study. Aldurazyme was digestedwith PNGase F or Endo H overnight and then subjected to theenzyme assay with 4-methylumbelliferyl α-L-iduronide (17) as thesubstrate. A small amount of each digested sample was subjectedto ConA-Sepharose pull-down assaying to detect the residualN-glycans. The PNGase F–treated hIDUA showed no defect inenzyme activity, and some N-glycans showed PNGase F resistance(Fig. 1B, lanes 10 and 12). These glycans became sensitive to

Author contributions: N.M. and H.S. designed research; N.M., T. Tsukimura, and T. Taniguchiperformed research; N.M., T. Tsukimura, T. Taniguchi, S.S., K.O., and H.T. analyzed data;and N.M., H.T., and H.S. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org (PDB ID codes 3W81 and 3W82).1To whom correspondence should be addressed. E-mail: nmaita@tokushima-u.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1306939110/-/DCSupplemental.

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PNGase F on denaturation (Fig. 1B, lane 8). These results suggestthat some N-glycans of hIDUA are so rigid or buried, and theycannot gain access to the catalytic site of PNGase F. We observed

that in the presence of Nonidet P-40, the activity increased byabout twofold (Fig. 1B, lanes 3 and 4). This result is presumablydue to the prevention of unfavorable aggregation of hIDUA (18).

Fig. 1. Deglycosylation study of hIDUA. (A) Re-action scheme of dermatan sulfate hydrolysis cata-lyzed by hIDUA. (B) hIDUA was deglycosylated withPNGase F overnight under the indicated conditions(lanes 1–6), and then enzyme activities were mea-sured (lanes 1–6, bottom; ND, not detected). Smallamounts of the reaction mixture were mixed withConA-Sepharose and washed three times, and thenConA-gel was separated by SDS/PAGE (lanes 7–12).The gel was stained with CBB. Nonidet P-40 pre-vents unfavorable aggregation, resulting in in-creasing activity. Human IDUA aggregates wereproduced due to the reaction mixture’s pH (20). (C)Deglycosylation analysis of hIDUA with Endo H. Theprocedure was the same as that in B other than theuse of Endo H.

Fig. 2. Crystal structure of IdoA-bound hIDUA. (A) Domain organization of hIDUA. N-glycosylation sites observed in the crystal structure are indicated asyellow hexagons. N336, another N-glycosylation site, but with no glycans, is also indicated. The disulfide bond between C541 and C577 is indicated as S-Sbelow the bar. The overall structure of hIDUA complexed with IdoA (side and end views) is shown. Human IDUA (subunit A) is shown as a cartoon repre-sentation with the TIM barrel domain (pink), β-sandwich domain (green), and Ig-like domain (blue). N-glycans (yellow), IdoA (blue), and phosphate are shownas stick models. The positions of N-glycosylation sites are indicated. (B) A stereo image of the omit map of the high mannose type N-glycan attached to N372(subunit A) in an apo-state crystal. The map is contoured at 2.5σ. N372 and catalytic glutamates (E182 and E299) are also shown. (C) Schematic drawing of theN-glycan structure at N372. The cleavage sites for Endo H and PNGase F are also indicated. Glycan linkage patterns are denoted as follows: β4, β1–4; α2, α1–2;α3, α1–3; α6, α1–6.

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On the other hand, on Endo H digestion, the hIDUA activity wasreduced to 7% (Fig. 1C, lane 5). Endo H–resistant N-glycans werealso observed (Fig. 1C, lanes 8 and 10); this is in agreement withthe previous study showing that hIDUA expressed in CHO cellscarries a complex type of N-glycans (8). These results suggest thathigh-mannose and/or hybrid type N-glycans have a rigid confor-mation and affect the activity of hIDUA.

Overall Structure of hIDUA. To determine the structural basis ofthe relationships between the N-glycans and enzyme activity ofhIDUA, we solved the crystal structures of apo- and IdoA-boundhIDUA at 2.3- and 2.76-Å resolution, respectively (19) (Fig. 2A;Table S1). We could build an almost full-length hIDUA (resi-dues 27–642). There are two hIDUA subunits per asymmetricunit. Judging from the small buried surface area (548.3 Å2

against the total accessible surface area of 25,127 Å2) and theresults of gel filtration analysis, these two subunits are unlikely torepresent a functional dimer (20). hIDUA consists of threedomains: residues 42–396 form a classic (β/α)8 triosephosphateisomerase (TIM) barrel fold, residues 27–42 and 397–545 forma β-sandwich domain with a short helix–loop–helix (482–508),and residues 546–642 form an Ig(Ig)-like domain. The latter twodomains are linked through a disulfide bridge between C541 andC577. The β-sandwich and Ig-like domains are attached to thefirst, seventh, and eighth α-helices of the TIM barrel. A β-hairpin(β12–β13) is inserted between the eighth β-strand and the eighthα-helix of the TIM barrel, which includes N-glycosylated N372(Fig. 2A). The topologies of the TIM barrel and β-sandwichdomains of hIDUA are almost identical to that of XynB, whichbelongs to the same GH family 39 (12, 13). However, XynB hasa shorter amino acid length than hIDUA and lacks the C-ter-minal Ig-like domain (Fig. S1).We observed at least five N-glycans in the electron density

map other than that at N336 (Table 1). The loop region in-cluding N336 exhibited slightly poor electron density, and wecould not place any sugars there. Although only one or twosugars were detected in most of the N-glycans, we observed longoligosaccharide chains at N190 (subunit B) and N372 (subunitsA and B). N190 has complex type oligosaccharides, as previouslypredicted (8). The GlcNAc3Man2Gal1Fuc1 structure (Fig. S2)was visible at N190 of subunit B. This oligosaccharide interactswith the 590–592 loop region of symmetry-related subunit B.The most remarkable feature is the N-glycan attached to

N372, which is of the high-mannose type (8), and we clearlyobserved a GlcNAc2Man8 oligosaccharide chain in subunit A(Fig. 2B). The N-linked oligosaccharide chain is tightly bound tothe surface of the TIM barrel. The tip of mannose residue (Man7)reaches the active site, and constitutes a part of the substrate-binding pocket (Fig. 2B). The N-glycan at N372 interacts with

a protein through many polar- and water-mediated contacts (Fig.S3) including the side chains of H58, W306, S307, and Q370 andthe backbone carbonyls of P54, L56, and H356. In addition,a hydrophobic interaction between Y355 and Man3 was ob-served. The structural characteristics of N-glycan at N372 werevery similar in both subunits. A similar N-glycan interaction withan enzyme has been reported for Trichoderma reesei β-galacto-sidase. In the structure of the latter, the tip of the N-glycan at-tached at N930 comes near the active site; however, it seems toofar for any direct interactions with a substrate (21).

Table 1. N-glycan structures observed in the IDUA crystal structures

Subunit Position apo-IDUA holo-IDUA

Subunit A N110 GlcNAc GlcNAcN190 Not observed Not observedN336 Not observed Not observedN372 (GlcNAc)2(Man)8 (GlcNAc)2(Man)8N415 (GlcNAc)2 (GlcNAc)2N451 Not observed Not observed

Subunit B N110 GlcNAc GlcNAcN190 (GlcNAc)2(Fuc)(Man)2(GlcNAc)(Gal) (GlcNAc)(Fuc)N336 Not observed Not observedN372 (GlcNAc)2(Man)7* (GlcNAc)2(Man)7*N415 (GlcNAc)2 (GlcNAc)2N451 GlcNAc Not observed

*Man9 was not observed.

Fig. 3. Substrate binding site of hIDUA for IdoA. (A) Molecular surfacerepresentation around the substrate-binding pocket of hIDUA (subunit B).Mannose and basic residues are colored yellow and blue, respectively. TheIdoA molecule and phosphate are drawn as stick models. An omit map of theIdoA (contour at 3σ) is also shown. (B) Details of the interactions betweenthe IdoA and hIDUA (subunit B) are shown as a stereo image. The hydrogenbonds between IdoA and other related residues are indicated by black andcyan dashed lines, respectively.

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Very recently, two crystal structures of hIDUA were releasedin the Protein Data Bank with the space groups of R3 (PDB IDcode 4JXO) and P21 (PDB ID code 4JXP). The structures alsocontain a high-mannose type of N-glycan chain at N372; how-ever, they have six sugars at most and lack Man7, possibly owingto the plant seed expression system used (22).

N-Glycan Is Involved in the Substrate Interaction. In the IdoA-boundhIDUA structure, we clearly observed the electron density of theIdoA molecule at the center of the TIM barrel (Figs. 2A and 3A).There is little structural difference between the apo and IdoA-bound forms (rmsd = 0.253 Å, over 580 Cα atoms); only the sidechain of D187 is flipped toward the active site, which forms hy-drogen bonds between the Oδ2 atom and the backbone oxygenand Nδ atom of N181 (Fig. S4A). This change presumably yieldsa tight hydrogen network between the O2 atom of IdoA and Nδof N181 (Fig. 3B).There are 19 polar contacts between IdoA and the protein and

2 more contacts between IdoA and Man7 of the N-glycan (Fig.3B; Table S2). The two oxygen atoms of the carboxyl group ofIdoA interact with the backbone amide between G305 and W306and the side chains of K264 and R363. The O4 atom of IdoAinteracts with D349 and R363. The O3 atom also interacts with

H91, D349, and Man7. In addition, the O2 atom interacts withH91, N181, and E299, a catalytic glutamate. The O1 atom in-teracts with Man7 and E182, another catalytic glutamate. Theholo-hIDUA structure also provides us with information aboutthe substrate specificity of the enzyme. For example, β-D-glucuronicacid, an epimer of IdoA, may have less affinity than IdoA forthe binding site, as the hydrogen bonding between O5 and K264Nζ found in IdoA (3.18 Å) will be lost in β-D-glucuronic acid(3.94 Å; Fig. S4B).The O1 atom of IdoA faces the open side of the binding

pocket, suggesting that downstream of the dermatan/heparansulfate chain stretches out of this side (Fig. 3A; Fig. S5). Weobserved a phosphate ion between the side chains of H185 andH226. A putative hIDUA and dermatan sulfate complex modelsuggests that the sulfate moiety of IdoA-2-sulfate at the +2 po-sition overlaps the phosphate (Fig. S5). Thus, H185 and H226presumably interact with the sulfate moiety of the substratesugar chain.

Amount of Glycan at N372 Correlates with the Enzyme Activity. Be-cause our structural study indicated that the N-glycan at N372interacts with an IdoA molecule, we focused on N372. Aldur-azyme was incubated with Endo H for varying times at 37 °C and

Fig. 4. Characterization of N-glycosylation at N372. (A) LC-MS profile of the native 369–383 peptide obtained on trypsin digestion of hIDUA. The spectra ofthe trivalent-ionized glycosylated (Upper) and deglycosylated (Lower) peptides are shown. The peak corresponding to m/z = 674.7 is magnified 100 times forclarity. (B) LC-MS profile of the Endo H–treated 369–383 peptide obtained on trypsin digestion of hIDUA, with the same representation as in A. The 1,050–1,300 range of the horizontal axis is magnified 1,000 times for clarity. (C) Ratio of the residual N-glycans during Endo H treatment determined by MS. Thedata were calculated using the peak area of peptide-GlcNAc2Man7–9 or peptide-GlcNAc of triply and quadruply charged ions. The enzyme activities of EndoH–treated (+Endo H) and nontreated (−Endo H) hIDUA are also plotted. (D) Michaelis-Menten plots for Endo H–treated (+Endo H, 48 h) and nontreated(−Endo H) hIDUA. The data are means of three repeated experiments ± SD. (Inset) Kinetic parameters of Endo H–treated and nontreated hIDUA.

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subjected to enzyme assay. We reduced the amount of Endo H tosee the effect of partial deglycosylation. The activity of hIDUAgradually decreased in an incubation time-dependent manner,the activity being almost completely lost after 48 h (Fig. 4C). Thekcat values of the native and deglycosylated (+Endo H, 48 h)hIDUA were 210 ± 3 and 9.50 ± 0.04 s−1, and the Km values were290 ± 10 and 180 ± 0.4 μM, respectively (Fig. 4D). To determinewhether the activity reduction was due to unfolding of the pro-tein, we measured circular dichroism spectra of the native anddeglycosylated hIDUA. The spectra were almost identical, sug-gesting that no aggregation or denaturation had occurred onEndo H treatment (Fig. S6).To clarify whether the Endo H treatment indeed caused

deglycosylation at N372, we digested the Endo H–treated hIDUAwith trypsin, followed by analysis by LC-MS. As a result, we clearlydetected triply and quadruply charged ions corresponding to theN372-containing fragment (residues 364–383) with GlcNAc andGlcNAc2Man7–9 (Fig. 4 A and B; Table S3). The ratio of thefragment (364–383) with GlcNAc2Man7–9 to the total fragment(364–383) was highly correlated with the enzyme activity (corre-lation: 0.989; Fig. 4C).Furthermore, we examined the amounts of the other N-gly-

cans attached to N336, N415, and N451, which could be cleavedby Endo H (8). The hIDUA treated with Endo H was digestedwith chymotrypsin and the peptide GlcNAcs, the Endo Hproducts, were monitored (Fig. S7; Table S4). The amounts ofthe peptide GlcNAc at N336, N372, N415, and N451 increased ina time-dependent manner. However, the degrees of the inversecorrelations between the enzyme activities and the amount ofN372-GlcNAc were higher (correlation: −0.915) than those forthe other N-glycosylation sites (N336, −0.616; N415, −0.615; andN451, −0.788) (Fig. S7). These results suggest that the N-glycanat N372, which forms a part of the substrate binding site, is in-volved in the enzymatic activity.

DiscussionOur structural and deglycosylation studies indicated that theN-glycan at N372 is essential for hIDUA activity. The deglyco-sylation study showed the different effects of Endo H andPNGase F (Fig. 1 B and C). The presence of PNGase F–resistantglycans was also observed in the previous study using CHO cell–expressed hIUDA (8). Furthermore, a report has described thattreatment of Aldurazyme with PNGase F decreased enzymeactivity by 50%, but significant activity still remained (23). Thecrystal structure revealed that the tightly bound oligosaccharidechain was linked to N372, which can be explained by the PNGaseF resistance of the N-glycan. The kcat and Km values of the WTand deglycosylated hIDUA suggested that deglycosylationaffects enzyme catalysis and substrate binding. As seen in the

classical GH-A clan, the O1 atom of IdoA is the target for hy-drolysis, and the carboxyl group of E182 would act as a protondonor to O1 (24, 25). We could observe the interaction betweenthe O1 atom of IdoA and the O3 atom of Man7 in subunit B(Fig. 3B; Table S2). Thus, Man7 may somehow influence pro-tonation of the O1 atom of IdoA.The results of a molecular phylogenetic analysis of IDUA

orthologs suggest the importance of the N-glycan at N372.Multiple sequence alignment showed that in all of the IDUAs,i.e., the Ciona to human ones, the positions corresponding toN372 and T374 are conserved (Fig. 5; Fig. S8), whereas fiveother N-glycosylation sites are not (Fig. S8). Additionally, iden-tical or similar residues are clustered at the reaction center andalong the interface of the N-glycan at N372 (Fig. S9A). Thesefindings strongly suggest not only the conservation of this as-paragine residue but also N-glycosylation throughout multicel-lular animals, and thus, the N-glycosylation at N372 should playa significant role in the function of IDUA.We could observe a phosphate ion around H185, H226, and

R230 (Fig. 3A; Fig. S5). These residues are conserved amongvertebrates (Fig. S8). The surface electrostatics showed thatthere is another positively charged patch, comprising H226 andR263, just outside the exit to the substrate binding cleft (Fig.S9B). These positively charged residues are also highly conservedamong vertebrates (Fig. S8). Such positively charged patches arelikely to contribute to binding of the sulfated glycosaminoglycanof dermatan/heparan sulfate. These results suggest that thevertebrate IDUAs have adapted to dermatan sulfate, the maincomponent of skin.More than 55 disease-associated missense mutations in the

human α-L-iduronidase (IDUA) gene have been identified (Hu-man Gene Mutation Database, www.hgmd.cf.ac.uk/). Among them,we paid attention to the W306 to Leu (W306L) gene (26) as a

Fig. 5. The loop inserted between β8 and α8 (348–384) is highly conserved. (A) The loop inserted between β8 and α8 (348–384) is colored magenta. N372 isdrawn as a stick model. (B) Alignment of sequences corresponding to the loop inserted between β11 and α10 (348–384) of human IDUA. Sequences werealigned using Clustal X ver.2 (34), and colored with ESPript (35). The names of the species and protein IDs are given in the legend to Fig. S8. N372 and T374 areindicated by pink arrowheads.

Fig. 6. Molecular modeling of the Trp306 to Leu mutant. (A) Polar contactbetween W306 and Man7. (B) Superposition of energy-minimized WT (cyan)and W306L (green). The side chains of W306 (Leu306), S307, and F352 areindicated as a stick model.

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possible mutation that affects the N-glycan at N372. Structuralanalysis revealed that W306 interacts with the N-glycan at N372,and we examined whether this mutation would lead to a dysfunctionof enzyme activity by molecular modeling and energy minimization.The rmsd between theWT andW306L is 0.023 Å, and the locationsof S307 and F352 in the IDUA molecule were predicted to moveslightly with the amino acid substitution (Fig. 6). This finding sug-gests that the amino acid substitution does not affect the catalyticcenter or, if at all, just a little. TheW306L mutation may cause MPSI through the effect on the conformation of the N-glycan at N372.Most crystallographers remove the glycans when they try to

crystallize glycoproteins to improve their homogeneity. However,the functionally important glycans tend to bind to a protein tightlyand are resistant to the processing in the endoplasmic reticulumand Golgi apparatus (21, 27–29). Our study suggests that it isbetter to retain the high-mannose type glycans to not overlook theessential functions.In conclusion, we first determined the structure of hIDUA,

and then structural and biochemical studies revealed that theN-glycan at N372 was used as a substrate-binding module. Fur-thermore, the results of a kinetic study suggested that theN-glycan is directly involved in enzyme catalysis. These findingswill be useful not only for elucidation of the molecular basis ofMPS I but also for the development of new drugs for this disease.

Materials and MethodsThe methods are described in full in SI Materials and Methods.

Samples and Chemicals. The recombinant hIDUA expressed in CHO cells(Aldurazyme) was purchased from Genzyme Japan. IdoA was purchasedfrom Carbosynth (United Kingdom).

Crystallization, Structure Determination, and Model Refinement. We crystal-lized and solved the structure of hIDUA by the single isomorphous replacementwith anomalous scattering (SIRAS) method as described previously (19). Weautomatically built an initial model using RESOLVE (30) and subsequently fixedit by hand with COOT (31), and then refined the apo-hIDUA structure with CNS(32) and REFMAC5 (33).

ACKNOWLEDGMENTS. We thank the beamline staff at the Photon Factoryand SPring-8 BL44XU for supporting the data collection under Proposals2009G074 and 2011G135. We also thank H. Saito, C. Mizuguchi, I. Sagawa(Tokushima University), T. Nishino (National Institute of Genetics), and M.Ariyoshi (Kyoto University) for supporting the data collection. This work wasperformed with a Cooperative Research Grant from the Institute for EnzymeResearch, Joint Usage/Research Center, University of Tokushima. This workwas supported by Grants-in-Aid for Young Scientists (20770085) and Scien-tific Research (23570139) from Japan Society for the Promotion of Science(to N.M.) and the Program for the Promotion of Fundamental Studies inHealth Sciences of the National Institute of Biomedical Innovation (ID 09-15) (H.S.).

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