INTRODUCTION

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Eukaryotic glycosylasparaginase (glycoasparaginase, N⁴(-N-acetyl-D-glucosaminyl)-L-asparaginase, 1-aspartamido--N-acetylglucosamine amidohydrolase, aspartylglycosylaminase, aspartylglucosaminidase, EC 3.5.1.26) is a well known lysosomal enzyme that cleaves the amide bond of asparagine-linked glycoproteins (1). It is widely distributed in vertebrate tissues (2) and insect cells (3) and is also found in bacteria (4). Substrate preferences for this enzyme include free -amino and -carboxyl groups on the asparagine, and that position 6 of N-acetylglucosamine does not contain fucose. However, a recent study suggests that the -amino and -carboxyl groups on the asparagine part of the substrate may not strictly be required for hydrolysis (5).

A deficiency of glycosylasparaginase (GA)¹ results in accumulation of glycoasparagines in tissue lysosomes and leads to severe clinical symptoms, known as aspartylglycosaminuria (AGU). AGU is the most common disorder of glycoprotein degradation. It severely involves the central nervous system and causes skeletal abnormalities and connective tissue lesions. Among children in eastern Finland, AGU was found to be the leading genetic cause for mental retardation after trisomy 21 and fragile X syndrome (1).

Glycosylasparaginase has been biochemically characterized from different species and is composed of two nonidentical subunits of approximately 24 and 20 kDa, associated by noncovalent forces. These respective subunits are referred to as the a- and b-subunits (or heavy and light subunits). The enzyme is encoded by a single gene, and post-translational cleavage of the nascent polypeptide into a mature a/b heterodimer is required for activation. Neither the single chain precursor (6, 7) nor the isolated subunits (8) are enzymatically active by themselves. Expression of the a- and b-subunits of GA on separate DNA constructs showed that independently folded subunits lack enzyme activity, and even when co-expressed in vitro they fail to produce an active heterodimer (9). A common feature of GA from different species is a new N-terminal threonine of the C-terminal product (the b-subunit) resulting from the autoproteolytic activation (10). A study demonstrated that an irreversible inhibitor specifically reacts with the N-terminal threonine on the b-subunit of the human leukocyte enzyme via an -ketone ether linkage with the hydroxyl side chain (8), indicating that this N-terminal threonine acts as a nucleophile during substrate hydrolysis. The crystal structure of human GA shows a topology similar to other N-terminal nucleophile hydrolases (11, 12) and reveals interactions between the N-terminal threonine and aspartate, one of the reaction products (13).

GA from Flavobacterium meningosepticum is the only prokaryotic homolog characterized so far. It differs from the human counterpart in several aspects: (i) sequence alignment of these two enzymes reveals only about 30% sequence identity and shows a difference in one gap/insertion of 31 residues (3, 4); (ii) part of the 31-residue insertion in the human enzyme is removed from the new C terminus of the a-subunit in the lysosome (6); no trimming occurs in the bacterial enzyme; (iii) the human enzyme contains N-linked glycans on both the a- and b-subunits (Asn¹⁵ and Asn²⁸⁵) (14), whereas the bacterial enzyme is nonglycosylated (4); (iv) according to previous sequence alignments (3, 4), neither the position nor the pattern of disulfide bridges is conserved between these two enzymes. The disulfide bonds have been shown to be essential for initial protein folding and activation of human GA (15). Together, these differences suggest the possibility that the structure of Flavobacterium GA may be significantly different from the human enzyme. Here we report the crystal structures of recombinant GA from F. meningosepticum. Structures of the wild type GA and a single amino acid mutant in their mature forms have been determined at 2.2 and 2.1 Å, respectively.

	MATERIALS AND METHODS

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Crystallization and Crystal Preparation-- Protein expression and purification will be described elsewhere.² Crystals were grown in hanging drops equilibrated by vapor diffusion against well solutions of 15% PEG 3300, 100 mM HEPES, pH 7.5, 0.1% sodium azide. Microseeding was routinely used to improve crystal quality. Crystals were harvested into a modified well solution containing 20% PEG 3300. Shortly before data collection, crystals were placed in dialysis buttons and dialyzed stepwise against harvest buffer supplemented with a progressively higher concentrations of glycerol. The final glycerol concentration was 20%, and the transfer steps were between 5 and 10% glycerol and varied from 3 h to a couple of days for each step. No correlation was observed between transfer procedure and the quality of the diffraction data. Heavy atom derivatives were obtained by soaking the native crystals in the harvest buffer supplemented with 10% glycerol and 0.1-10 mM of heavy atom compound, from 15 h to a few days, before step up to the final glycerol concentration.

Data Collection and Processing-- Oscillation data were collected from crystals frozen at 100 K, mounted in a thin film of harvest buffer plus 20% glycerol, and supported by a loop made of dental floss. Diffraction data were collected using an R-AXIS IIC image plate detector mounted on a Rigaku RU300 rotating anode generator. All intensity data were processed and scaled using the programs DENZO and SCALEPACK (16) and converted to structure factors using TRUNCATE from the CCP4 software package (17). The space group was determined by examination of the differences in intensities of potential pairs of reflections across putative mirror planes within the data and confirmed by examining the electron density maps.

Experimental Phases-- Heavy atom positions were initially obtained from isomorphous Patterson maps calculated in XTALVIEW (18). The heavy atom parameters were then refined by MLPHARE from the CCP4 package (17) and were confirmed by the cross Fourier. At this stage, the figure of merit was 0.55 (0.71 calculated by XTALVIEW). The MIR phases were then extended to the resolution range 12-2.5 Å and were improved by noncrystallographic symmetry averaging, solvent flatterning, and histogram matching using the program DM (17) with a figure of merit of 0.785.

Model Building and Refinement-- The first map was calculated at 2.5 Å resolution (see Fig. 3) and skeletonized to build a C trace in the program O (19). The first 244 of 275 residues were built based on the DM-modified MIR map. Automated refinement included rigid body, overall temperature factor, positional, and restrained atomic temperature factor refinement, as well as simulated annealing using a slow cooling protocol in X-PLOR (20). After the first round of manual rebuilding, without the N-terminal nucleophile (amino acid 152), the structure, after rigid body fit by AMORE (17), was used for refinement of both the wild type and the T152C mutant. SIGMAA (17) was used in the early cycles of refinement and manual rebuilding to combine model phases with experimental phases. Initially, strict noncrystallographic symmetry constraints were applied, and in later stages of refinement, tight noncrystallographic symmetry restraints were applied, exclusive of residues that were involved in crystal contacts. After a few rounds of model rebuilding, stepwise resolution extension, and automated refinement, clear electron density could be seen for all residues in the final model. Refinement protocols were aimed at decreasing the R_free (21) rather than the conventional R_cryst to avoid errors introduced by overfitting of the data. When the R_free appeared to have reached a minimum at the final resolution, water molecules were added and subjected to another round of automated refinement and manual rebuilding. The statistics of the final structures are shown in Table II, with a root mean square (r.m.s.) deviation of 0.20-0.23 Å for main chain atoms between crystallographically independent molecules.

Structural Comparisons-- All superimpositions of different structures were performed using LSQKAB (17). For Fig. 4, all atoms of residues contacting the reaction product, aspartate, in the human structure are superimposed (atoms equivalent to those in bacterial Thr¹⁵², Thr¹⁷⁰, Arg¹⁸⁰, Asp¹⁸³, Thr²⁰³, and Gly²⁰⁴).

	RESULTS

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Description of the Structure-- The enzymes crystallized in space group P2₁ with unit cell constants a = 46.2Å, b = 97.3Å, c = 61.8Å, and  = 90.3°. The initial phases were obtained by MIR method with four heavy atom derivatives (Table I). The wild type structure has been determined at 2.2 Å and refined to an R_free (21) of 29.70% and an R_cryst of 24.65% with all reflections (Table II). There are two a/b heterodimers per asymmetric unit. In the final model, each heterodimer comprises 136 residues (3-138) of the a-subunit and 139 residues (152-290) of the b-subunit. No electron density is observed for the 13 residues spanning the segment (139-151) that connects the a- and b-subunits in the precursor protein. In the crystal, this linker segment appears to face into the solvent channels. 93% of the nonglycine residues fall in the most favored regions of Ramachandran plot, as defined in PROCHECK (22), and no residues are in the disallowed regions.

View larger version (42K): [in this window] [in a new window]   Fig. 1.   The structure of glycosylasparaginase from F. meningosepticum. a, stereo ribbon representation of the Flavobacterium GA structure. One heterodimer is shown with a- (red) and b-subunits (green). The active site is at top center of the structure toward the viewer and around the N-terminal end of the b-subunit (green) (labeled Nb in light blue). b, stereo diagram of C traces of Flavobacterium GA. c, stereo diagram of C traces of Flavobacterium (dark blue) and human (gray) GA. Superimposition is based on all common main chain atoms (excluding insertions/deletions). The residues labeled are in Flavobacterium sequence number.

View larger version (54K): [in this window] [in a new window]   Fig. 2.   Structure-based sequence alignment between the Flavobacterium (Flavo) and human glycosylasparaginase (Human). The dashed lines represent incorporated gaps that bring the sequences into alignment. The vertical lines represent identical matches. The bold black arrow represents the autoproteolytic site. Yellow shading shows conserved residues in the active site (see Fig. 4 and text). Purple shades highlight the mutations identified in human AGU disease (1); not shown is an Asp-Ala insertion between Ala19 and Ala20 in the human sequence. The secondary structural elements are based on this study: red arrows for -strands, green rectangles for -helices, and open rectangle for 310 helix. Cysteine residues forming disulfide bonds in the human structure are linked by lines below the human sequences: Cys41-Cys46, Cys140-Cys156, Cys263-Cys283, and Cys294-Cys322.

Structural Differences between Flavobacterium and Human Structures-- An r.m.s. deviation of 1.4 Å is obtained by superimposing the common 1,068 main chain atoms (excluding insertions/deletions) of the Flavobacterium and human GA structures. This is significantly larger than the r.m.s. deviation of 0.26-0.45 Å found between the two human structures (13). Moreover, the r.m.s. deviation between our two bacterial structures is 0.22 Å (see below), similar to that observed between two heterodimers in the asymmetric unit (Table II). A number of peptide fragments within the structure deviate by more than 2 Å (Fig. 1c); most of them are in loops connecting elements of secondary structure. The largest difference of 8.5 Å is near the 7-residue insertion in the bacterial structure. Deviations greater than 2 Å are also observed in the common secondary structural elements (see below). These data are consistent with the observation that molecular replacement using the human structure proved difficult with the data of Flavobacterium GA.³

View larger version (54K): [in this window] [in a new window]   Fig. 3.   Quality of experimental map at 2.5 Å obtained with MIR phases modified using the program DM (17). The map is contoured at 1.1 . The final model of the C-terminal end of -helix aH1 is shown by atom type: yellow for carbons, blue for nitrogens, and red for oxygens. The best fit structure of human GA based on the common secondary structural elements is also superimposed (shown in gray) to visualize the differences.

Active Site and Mechanism-- The loops connecting different layers of -helices and -sheets form a deep funnel-shaped active site centered at the N-terminal Thr¹⁵² of the b-subunit (Fig. 1). The funnel in the bacterial enzyme is wider than that of the human enzyme, mainly because of deviation of the loop between helix aH2 and strand aS2 as well as lack of the C-terminal loop in the a-subunit (Fig. 1c). Several conserved residues surround the nucleophilic center Thr¹⁵² of the bacterial active site, including Thr¹⁷⁰, Arg¹⁸⁰, Asp¹⁸³, Thr²⁰³, and Gly²⁰⁴, which are highlighted in yellow in Fig. 2. These residues had been described to interact with aspartate, one of the two reaction products (13). As depicted in Fig. 4a, the human equivalent to Arg¹⁸⁰ forms hydrogen bonds with the -carboxyl group of aspartate. Both human equivalent residues of Asp¹⁸³ and Gly²⁰⁴ make hydrogen bonds with the -amino group of aspartate. Human residues equivalent to Thr¹⁵², Thr²⁰³, and Gly²⁰⁴ also form hydrogen bonds with the O1 of aspartate. In addition, human residue equivalent to Thr¹⁷⁰ makes a hydrogen bond with the O of Thr¹⁵².

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Stereo view of the active site of glycosylasparaginase. a, stereo view of superimposition of the active sites between Flavobacterium (shown according to atom type: yellow for carbons, blue for nitrogens, and red for oxygens) and human (shown in gray) GA. Also shown is aspartate in the human enzyme/product structure (13). Dashed lines correspond to the hydrogen bonds described in the human structure. b, the same stereo view of active site in the T152C mutant. The color scheme is the same as the wild type in (a), except that sulfur of the thiol group is shown in green.

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Structure of the T152C Mutant-- Thr¹⁵² plays a key role in catalysis (4, 7, 8). Substitution of the N-terminal nucleophile Thr¹⁵² by a thiol group (T152C mutant) reduces k_cat by 5 orders of magnitude (23). Autoproteolysis in this mutant is also very slow but can be accelerated by hydroxylamine (10). In this study, we have also determined the three-dimensional structure of the T152C mutant in its mature form at 2.1 Å and refined to an R_free of 28.06% and an R_cryst of 23.32% with all reflections (Table II). The structure of this mutant is essentially identical to that of the wild type enzyme with an r.m.s. deviation of 0.22 Å for all the main chain atoms and 0.25 Å for the active site atoms (Fig. 4b). This indicates that the reduction of reaction rate of this mutant is because of the change of chemical groups at the side chain of residue 152.

Quaternary Structure of GA-- Bacterial GA forms a dimer of a/b heterodimers in the crystals (Fig. 5a). A similar quaternary structure is also observed in the crystals of human GA in different crystal packings (13). The surface interactions between pairs of heterodimers are extensive and mainly involve hydrogen bonds and hydrophobic contacts (Fig. 5, b and c). Basically, both heterodimers use the same hydrophobic surface for the (a/b)₂ tetramer formation, reminiscent of hand shaking. The main interface interactions come from the strand aS4, helix bH2, and the loops between aH3 and aS4, aS4 and aH4, bS2 and bS3, and bH1 and bH2 in both heterodimers. In addition, the bacterial enzyme has unique interactions between the 7-residue insertion and the loop connecting -strands aS2 and aS3. The human structure has substantially more interactions from the unique C-terminal loop of the a-subunit. Dimerization of a/b heterodimers sequesters a solvent-accessible surface area of 1882 Å² from each a/b heterodimer of the bacterial GA, compared with 2485 Å² for the human enzyme. The smaller interface and thus weaker dimer interaction between two heterodimers of bacterial GA may explain why no (a/b)₂ tetramers of the bacterial GA are observed on sizing columns.⁵ Nonetheless, the existence of bacterial (a/b)₂ tetramers in the crystals suggest that the heterotetramers of bacterial GA may also exist in solution. This heterotetrameric structure of (a/b)₂ has been observed in solution for the human (8), chicken (2), and insect GA (3).

View larger version (82K): [in this window] [in a new window]   Fig. 5.   Dimer structure of GA a/b heterodimers from F. meningosepticum. a, ribbon drawing of the quaternary structure of (a/b)2 heterotetramer. One heterodimer is in red (a-subunit) and green (b-subunit), and the other is in blue (a-subunit) and orange (b-subunit). The site of enzyme activity (and putative site of autoproteolysis) in each heterodimer is pointed out by an arrow. The view orientation is similar to that of Fig. 2 in Ref. 13. b, surface potential representation of the dimerization interface of the left heterodimer in a, produced using Grasp (39). The orientation has been rotated 90 ° to the left along the vertical axis in a. c, surface potential representation of the dimerization interface of the right heterodimer in a, produced using Grasp (39). The orientation has been rotated 90 ° to the right along the vertical axis in a.

	DISCUSSION

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Aspartylglycosaminuria-- The physiological importance of the glycosylasparaginase is revealed by the occurrence of a human genetic disorder, known as AGU, because of a deficiency of this lysosomal hydrolase (1). Many mutations in the GA gene that cause AGU have been reported, and more are likely to be found. However, a major obstacle to studying the consequences of these mutations is the difficulty to obtain recombinant human enzyme in sufficient quantities (28). In this study, four known AGU single mutations have been mapped onto the shared secondary structural elements between the bacterial and human enzymes (Fig. 2). A double mutant (human Arg¹³⁸ to Gln and Cys¹⁴⁰ to Ser) maps outside of the secondary structural elements and appears to result from the loss of a disulfide bond (Cys¹⁴⁰-Cys¹⁵⁶) that stabilizes a unique loop in the human enzyme. Thus, our work confirms the suitability of the bacterial enzyme as a model to analyze the consequences of mutations in AGU patients at the molecular level.

Structural Comparisons-- Glycosylasparaginase belongs to a newly classified family of enzymes that have a novel N-terminal threonine, serine, or cysteine that provides the nucleophile in their reaction mechanism (11). Previously reported structures of this family include glutamine 5-phosphoribosyl-1-pyrophosphate amidotransferase from Bacillus subtilis (29), Escherichia coli penicillin amidohydrolase (30), the 20 S proteasome from the archaebacterium Thermoplasma acidophilum (31) and yeast (32), human glycosylasparaginase (13), and the glutaminase domain of E. coli glucosamine 6-phosphate synthase (27). All of these enzymes have a similar protein fold comprised of a sandwich of antiparallel  sheets surrounded on either side by layers of  helices. Many of these enzymes are activated by cleavage of the peptide bond to free the -amino group to form the N-terminal nucleophile (10). A different protein fold has recently been described for the autoprocessing domain of Drosophila Hedgehog protein (33). It is an all  structure that is distinct from the GA structure but is related to the intein domain of PI-SceI endonuclease (34, 35).

Enzyme Mechanism-- Crystal structures described in this study, on the other hand, also raise questions about the detail mechanism of GA. Structure of the wild type GA from F. meningosepticum in its mature form has been determined at 2.2 Å resolution. Although the topology of the bacterial enzyme is very similar to that of the human structure, several significant differences have been observed. The active site of Flavobacterium GA is in an open conformation, whereas the human enzyme adopts a closed conformation that grasps the reaction product, aspartate (Fig. 4a). Moreover, the side chain of Thr²⁰³ may not be as important in stabilizing the negative oxyanion intermediate as previously suggested (13). This is consistent with a mutagenesis study in which replacement of Thr²⁰³ by Ala (T203A mutant) in the bacterial enzyme does not dramatically decrease the reaction rate (23). A three-dimensional structure of the enzyme-substrate complex is necessary to clarify the role of Thr²⁰³ side chain in the enzymatic mechanism.

Autoprocessing for Enzyme Activation-- Cis-autoproteolysis involves the intramolecular catalytic cleavage of a peptide bond and is required to activate many enzymes (12). In addition to GA, these include penicillin acylase (30), proteasomes (31, 36, 37), as well as the hedgehog family of eukaryotic developmental regulatory proteins (38). Autoproteolytic cleavage also serves as a mechanistic component for protein splicing (35). In contrast to the activation of zymogens, such as chymotrypsinogen and trypsinogen through proteolysis by another trypsin molecule, the autoproteolysis of GA is an intramolecular reaction (10). Human GA is also believed to undergo autoproteolysis to form the active enzyme but with some differences. First, the disulfide bridges in the human enzyme are essential for early folding and for autoproteolytic processing (15). In contrast, there are no disulfide bridges in the bacterial enzyme. Furthermore, part of the 31-residue insertion in the human enzyme is removed from the C terminus of the autoproteolyzed a-subunit in the lysosome by a second cleavage (6). No such trimming occurs for the bacterial enzyme.