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Crystal structure of a tetrameric GDP-D-mannose 4,6-dehydratase from a bacterial GDP-D-rhamnose biosynthetic pathway

¹ Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319, USA
² Canadian Bacterial Diseases Network, Department of Microbiology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada
³ Procter & Gamble Co., Miami Valley Laboratories, Cincinnati, Ohio 45252, USA

Reprint requests to: R. Michael Garavito, Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319, USA; e-mail: garavito{at}msu.edu; fax: (517) 353-9334.

	Abstract

TOP Abstract Introduction Results and Discussion Materials and methods References

 
D-Rhamnose is a rare 6-deoxy monosaccharide primarily found in the lipopolysaccharide of pathogenic bacteria, where it is involved in host–bacterium interactions and the establishment of infection. The biosynthesis of D-rhamnose proceeds through the conversion of GDP-D-mannose by GDP-D-mannose 4,6-dehydratase (GMD) to GDP-4-keto-6-deoxymannose, which is subsequently reduced to GDP-D-rhamnose by a reductase. We have determined the crystal structure of GMD from Pseudomonas aeruginosa in complex with NADPH and GDP. GMD belongs to the NDP-sugar modifying subfamily of the short-chain dehydrogenase/reductase (SDR) enzymes, all of which exhibit bidomain structures and a conserved catalytic triad (Tyr-XXX-Lys and Ser/Thr). Although most members of this enzyme subfamily display homodimeric structures, this bacterial GMD forms a tetramer in the same fashion as the plant MUR1 from Arabidopsis thaliana. The cofactor binding sites are adjoined across the tetramer interface, which brings the adenosyl phosphate moieties of the adjacent NADPH molecules to within 7 Å of each other. A short peptide segment (Arg35–Arg43) stretches into the neighboring monomer, making not only protein–protein interactions but also hydrogen bonding interactions with the neighboring cofactor. The interface hydrogen bonds made by the Arg35–Arg43 segment are generally conserved in GMD and MUR1, and the interacting residues are highly conserved among the sequences of bacterial and eukaryotic GMDs. Outside of the Arg35–Arg43 segment, residues involved in tetrameric contacts are also quite conserved across different species. These observations suggest that a tetramer is the preferred, and perhaps functionally relevant, oligomeric state for most bacterial and eukaryotic GMDs.

Keywords: GDP-D-mannose 4,6-dehydratase; deoxyhexose biosynthesis; short-chain dehydrogenase/reductase; low-barrier hydrogen bonds; lipopolysaccharide biosynthesis

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03393904.

	Introduction

TOP Abstract Introduction Results and Discussion Materials and methods References

 
The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen that causes infection in patients with impaired immune systems such as burn wound victims, cancer patients, and especially those with cystic fibrosis. Because of its intrinsic resistance to many antibiotics, infections by this bacterium are difficult to treat; therefore, efforts have been made to understand the host–bacterium interactions and the establishment of infection. The lipopolysaccharide (LPS), which plays a significant structural role in the outer membrane, contributes to P. aeruginosa virulence (Cryz et al. 1984). In fact, studies have shown that the LPS contributes to the establishment of chronic infections in patients with cystic fibrosis (for review, see Rocchetta et al. 1999). P. aeruginosa contains two variant forms of LPS known as A-band and B-band. B-band is a heteropolymer composed of many different monosaccharides, whereas A-band LPS is a homopolymer of D-rhamnose (for review, see Rocchetta et al. 1999).

The deoxyhexose sugar rhamnose is found in glycoconjugates of plants and bacteria but not in animals. D-Rhamnose is the rarer of the two isoforms, found only in the outermost cell surface components of bacteria. The source of D-rhamnose comes from the nucleotide-activated GDP-D-rhamnose, which is synthesized in two steps. The enzyme GDP-D-mannose 4,6-dehydratase (GMD) catalyzes the conversion of GDP-D-mannose to the intermediate GDP-4-keto-6-deoxy-D-mannose. This serves as a branching point to several different deoxyhexoses, including GDP-D-rhamnose, GDP-L-fucose, GDP-6-deoxy-D-talose, and the GDP-dideoxy amino sugar GDP-D-perosamine (Fig. 1). In the GDP-D-rhamnose synthesis, a reductase (RMD) that targets the 4-keto group has been identified in P. aeruginosa (for review, see Rocchetta et al. 1999) and Aneurinibacillus thermoaerophilus (Kneidinger et al. 2001). The enzymes involved in the GDP-L-fucose pathway have been identified in bacteria, plants, and animals, in which fucose is found in glycoconjugates. Fucose affects nodulation in rhizobial organisms (Lopez-Lara et al. 1996; Mergaert et al. 1997) is important in stem development and strength in plants (Bonin et al. 1997) and plays a role in human immune regulation (Varki 1994). Enzymes involved in the GDP-6-deoxy-D-talose and GDP-D-perosamine pathways have been identified in bacteria, in which both talose and perosamine are components of LPS of bacteria and perosamine plays a role in the glycosylation of antibiotics (Albermann and Piepersberg 2001; Maki et al. 2003). The involvement of GMD as the initiator enzyme in all these pathways makes it a particularly interesting target of research.

View larger version (16K): [in this window] [in a new window]   Figure 1. The biosynthetic pathway of several different deoxyhexoses originating from the same 4-keto-6-deoxy intermediate. GMD converts GDP-D-mannose to the common intermediate, which undergoes reduction, epimerization, or amination, resulting in a variety of deoxy sugars.

Somoza et al. (2000) determined the three-dimensional structure of GMD from E. coli and confirmed the structural relationship of GMD to the NDP-sugar modifying subfamily of SDR enzymes. However, their work indicated that GMDs exist primarily as an active dimer. In contrast, Mulichak et al. (2002) showed that the structure of the MUR1 isoform of GMD from Arabidopsis thaliana was tetrameric, and that the NADP(H) binding site was intimately involved in creating the tetramer interface. The question thus arises as to whether bacterial GMDs differ from eukaryotic GMDs in terms of their active oligomeric state and, perhaps, the regulation of enzyme activity. We report here the crystal structure of a GMD from the bacterium P. aeruginosa in the presence of the ligands NADPH and GDP. Our analysis again confirms the structural homology with the GMDs from all three species but reveals that the GMD from the bacterium P. aeruginosa exists as a MUR1-like tetramer. Based on the conservation of subunit interactions and sequence in GMDs, evidence indicates that the tetrameric form of this enzyme may be its active oligomeric state in both prokaryotes and eukaryotes.

	Results and Discussion

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Overall structure
As is characteristic of members of the NDP-sugar modifying SDR subfamily, the GMD monomer folds into two domains: the N-terminal cofactor-binding domain and the C-terminal substrate-binding domain (Fig. 2). The larger N-terminal domain consists of an alternating / motif of seven -strands in the order 3-2-1-4-5-6-7 flanked by -helices, yielding a modified Rossmann fold, an element generally associated with dinucleotide binding. Common in this subfamily is the transition into the C-terminal domain after 6, providing an extension of an -helix and two -strands. The chain then turns back to the N-terminal portion, adding another -helix and the seventh N-terminal -strand before returning to and completing the C-terminal domain. The smaller domain consists largely of three -helices with two sets of mixed parallel and antiparallel -sheets. The cleft that exists between the two domains is the site of dinucleotide cofactor and nucleotide-sugar substrate binding and is where catalysis occurs.

View larger version (72K): [in this window] [in a new window]   Figure 2. Ribbon representation of the GMD monomer (A), the GMD dimer highlighting the tetramer interface (B), and the GMD tetramer (C). For each drawing -strands are colored blue, and the areas colored in yellow represent the regions involved at the tetramer interface. In the active site of the GMD monomer and dimer, the cofactor NADPH and substrate GDP are shown in backbone representation. Only the cofactor NADPH is included in the tetramer for clarity. Arrows in the GMD dimer indicate the RR loops, and the asterisk highlights the close approach of the cofactors from each monomer to one another. The asterisks in the tetramer highlight the four-helix bundle, the dimerization mode common among SDR enzymes.

The tetramerization of GMD results in the adjoining of the cofactor binding sites at the interface such that the adenosyl phosphate moieties of bound NADPH molecules fall within 7 Å and 7.5 Å, respectively, for GMD and MUR1. Both GMD and MUR1 share a flattened ellipsoidal shape (~95 x 75 x 60 Å and ~100 x 74 x 57 Å, respectively). In contrast, tyvelose epimerase, which oligomerizes along the analogous surface, forms a less compact tetramer (~100 x 110 x 60 Å). Consequently, the tetramer interface of tyvelose epimerase is less extensive, and the adenine rings of bound NAD molecules are separated by 11 Å.

Cofactor binding site
There is well-ordered electron density in the active site to unambiguously place an NADPH molecule in each monomer. The nonplanar electron density corresponding to the nicotinamide ring indicates that the reduced form of the cofactor is present. NADPH is found in the cofactor-binding sites of the crystals when no cofactor was added to the crystallization conditions, indicating tight binding of the cofactor throughout purification. Attempts to replace NADPH with excess NADP or NAD(H) have so far been unsuccessful. A schematic diagram of the interactions of NADPH with the surrounding residues is shown in Figure 3.

View larger version (22K): [in this window] [in a new window]   Figure 3. Schematic diagram of the potential hydrogen bonds between the cofactor NADPH and substrate GDP-D-mannose with GMD. Mannose was modeled in using NADPH/GDP-D-rhamnose/MUR1 complex (1N7G [PDB] ) as a guide.

The nicotinamide moiety is bound in the syn conformation and may be stabilized in this orientation by hydrogen bonding between the carboxyamide nitrogen and the pyrophosphate. This conformation is consistent with other SDR enzymes, allowing a B-side hydride transfer during catalysis. In GMD the conformation may be further stabilized by hydrogen bonds between the carboxyamide group of the nicotinamide moiety to the main-chain amide nitrogen of His180 and the Arg185 side chain. The nicotinamide ribose hydroxyls are within hydrogen bonding distance to the catalytic residues Tyr150 and Lys154, interactions that are highly conserved in SDR enzymes.

The adenine ring of the cofactor is largely coordinated by the negatively charged side chain Asp59, which makes a potential hydrogen bond to the adenosyl amino group, whereas the main-chain amide nitrogen of the subsequent residue, Met60, hydrogen bonds to the N1 nitrogen of the adenine group. Both interactions are highly conserved among SDR enzymes. Further coordination of the ring nitrogen atoms occurs through hydrogen bonding with water molecules in the adenine ring pocket. The Arg36 side-chain hydrogen bonds to the 2'-phosphate group via both NE and NH1 nitrogen atoms. The position of this arginine is conserved among GMDs and other NADP-binding SDRs such as GFS, and is suggested to be responsible for discriminating between NADP and NAD (Somers et al. 1998). The hydroxyl of the adenosyl ribose is coordinated by the Thr11 side chain and by the main-chain amide nitrogen of Gly12. The main-chain amide nitrogen of Ala83 is within hydrogen bonding distance of the ribose ring oxygen. Similar interactions with the adenosyl ribose are seen in MUR1 and GFS (Somers et al. 1998; Mulichak et al. 2002).

Most intriguing about the cofactor binding site is the involvement of the adenosyl end in the tetramer interface, a feature also seen in the plant homolog MUR1 (Mulichak et al. 2002). The RR loop, a segment of nine residues (Arg35–Arg43), stretches into the neighboring monomer making not only protein–protein interactions but also contacts to the neighboring cofactor. Protein–protein interactions include Arg35 hydrogen bonding to Ser85* and Glu188*, Ser38 to Trp42*, and Arg43 to both Ser37* and Ser38*, as well as to the main-chain carbonyl of Ser38*. Protein–cofactor interactions include hydrogen bonding of Ser37 to the neighboring adenosyl 2'-phosphate via the main-chain nitrogen atom as well as the side-chain hydroxyl group. Arg35 also hydrogen bonds to the pyrophosphate of the neighboring NADPH, further tethering the tetramer together. Furthermore, the Arg36 side chains from each monomer are involved in a parallel stacking arrangement between the two adenine rings. They also help to coordinate the 2'-phosphate of their own NADPH in a way similar to that observed in GFS and MUR1.

A question raised by the participation of the cofactor in maintaining the tetramer interface is what does the GMD structure look like when NAD⁺ is bound. The plant homolog MUR1 (Mulichak et al. 2002) can use either NADP⁺ or NAD⁺ in the in vitro reaction (W.-D. Reiter, pers. comm.). Preliminary studies using a capillary electrophoresis (CE)-based assay have demonstrated that recombinant P. aeruginosa GMD, whether freshly purified or frozen for >1 year, readily converted GDP-D-mannose into product (data not shown). P. aeruginosa GMD is able to use both cofactors as evidenced by the nearly identical rates of substrate conversion observed when either NADP⁺ or NAD⁺ was present. However, as noted by Mulichak et al. (2002) and Somoza et al. (2000), the NADP(H) cofactor is quite difficult to remove. Hence, further characterization is needed to understand why both NADP⁺ and NAD⁺ support the GMD reaction in vitro, but GMDs can retain NADP(H) through purification.

Substrate binding site
The electron density corresponding to the substrate reveals that GDP is present in the active site of each monomer. Attempts to replace GDP with GDP-D-mannose by adding excessive amounts of the natural substrate to the crystallization conditions have not allowed us to successfully observe the hexose moiety. Instead, four water molecules apparently occupy the area corresponding to the position of the sugar. Of course, possible explanations for our observation are that there is a mixture of ligands in the substrate binding site, for example, GDP and GDP-D-mannose, or that under the current crystallization conditions, the mannose is disordered.

The mannose moiety could be modeled in using the NADPH/GDP-D-rhamnose/MUR1 (PDB code 1N7G [PDB] ) complex as a guide. Hydrogen bonding and van der Waals interactions were optimized while holding the GDP moiety constant. Binding interactions of the GDP-D-mannose with GMD are depicted in Figure 3. The orientation of the O6 hydroxyl of the GDP-D-mannose was chosen based on its ability to make potential hydrogen bonds to Thr126, Ser127, and Glu128. In the crystal structure, the presence of a water molecule close to the GDP-D-rhamnose C5 atom that makes hydrogen bonds to Ser127 and Glu128 further supports this proposed rotamer. Based on this model, both catalytic residues Thr126 and Tyr150 could hydrogen bond to the hexose O4 hydroxyl. Further coordination of the O2 and O3 hydroxyls and O5 of the hexose ring occurs through the side chains of Arg185, Tyr150, and the main-chain carbonyl oxygen of Ser85 and through water mediated hydrogen bonds.

The GDP moiety, which is completely buried in the small domain, is in the syn conformation. This is an unusual conformation for this nucleotide and may be related to substrate recognition. GDP is stabilized in this orientation by an intramolecular hydrogen bond between the guanine amine nitrogen atom and the phosphate. The pyrophosphate bridge of the GDP moiety abuts against the positive dipole of 6, in a manner similar to that of dinucleotide binding in a Rossmann fold. This allows for hydrogen bonding to the main-chain amide nitrogen of Val190. Further contacts to the GDP moiety include Asn179, Lys193, Arg218, Arg279, and Glu282, all highly conserved residues among GMDs. The ability of Arg279 to hydrogen bond to both phosphates is a feature conserved among several SDRs, although the exact position of the arginine is not conserved. Furthermore, the hydrogen bonding of Glu282 to the ribose hydroxyls is also observed in UDP-binding SDRs (Thoden et al. 1996c; Mulichak et al. 1999).

Catalytic mechanism
The proposed catalytic mechanism for GMD occurs via a three-step process (Fig. 4; Oths et al. 1990). The first step involves a hydride transfer from the mannose C4 to the C4 of the cofactor. An active site base deprotonates the O4 hydroxyl to yield the GDP-4-ketomannose intermediate (Fig. 4b). The second step involves the elimination of water from C6 to yield the GDP-4-keto-5,6-ene intermediate (Fig. 4c). In the final step, the C5–C6 double bond is reduced as the cofactor returns the hydride to the C6 position yielding GDP-4-keto-6-deoxy-D-mannose (Fig. 4d). The SDR enzymes share only a few conserved residues that include the catalytic triad Tyr-XXX-Lys and Ser/Thr, which are important in catalysis (Fig. 5; Persson et al. 1991). In addition, a highly conserved Glu among dehydratases has proven to be important in the dehydration step (Somoza et al. 2000; Allard et al. 2002). Mutagenesis and kinetics studies on E. coli GMD (Somoza et al. 2000) as well as other SDR enzymes have supported the roles of Tyr, Lys, Ser/Thr, and Glu in the reaction mechanism. The GMD reaction resembles that of other nucleotide hexose dehydratases, such as CDP-D-glucose 4,6-dehydratase and dTDP-D-glucose 4,6-dehydratase, that have been more extensively studied (He et al. 1996; Gross et al. 2000; Gerratana et al. 2001; Gross et al. 2001; Hegeman et al. 2001, Hegeman et al. 2002).

View larger version (25K): [in this window] [in a new window]   Figure 4. The mechanism proposed for GDP-D-mannose 4,6-dehydratase. The process can be divided into three steps: oxidation of GDP-D-mannose (A) to produce the 4-keto intermediate (B), dehydration into the 4-keto-5,6-ene intermediate (C), and, finally, reduction to yield GDP-4-keto-6-deoxy-D-mannose (D).

View larger version (72K): [in this window] [in a new window]   Figure 5. The alignment of GMD genes from various species. Conserved residues are highlighted in blue; similar residues are highlighted in light blue. Secondary structural elements are marked according to P. aeruginosa GMD. Residues involved in the dimer interface are boxed in copper; residues involved in the tetramer interface are boxed in yellow. An asterisk marks catalytic residues Thr126, Glu128, Tyr150, and Lys154.

To complete the oxidation step, an active site Tyr removes a proton from the O4 hydroxyl, forming the 4-keto intermediate. The catalytic Tyr150 in GMD is in proper position (distance of 2.7 Å) relative to the mannose model for it to directly attack the O4 hydroxyl. Early studies of E. coli GalE (Thoden et al. 1996b) and dTGDH (Allard et al. 2001) showed that the distance of Tyr to the O4 hydroxyl is too great for it to act directly as the base. The Ser/Thr catalytic triad member had been proposed to act as a proton shuttle to complete the oxidation step. However, more recent crystallographic studies of human GalE (Thoden et al. 2000), dTGDH (Allard et al. 2002), SQD1 (Mulichak et al. 1999), and MUR1 (Mulichak et al. 2002) showed that Tyr is within proper hydrogen bonding distance to directly attack the O4 hydroxyl. The Ser/Thr instead may orient the substrate in the active site and/or facilitate proton transfer. The presence of hydrogen bonds between the O4 and O6 hydroxyl and the Thr126 hydroxyl indicates that both these roles may be accomplished. To further facilitate the oxidation step, the catalytic Lys may stabilize Tyr in its negatively charged state. Studies indicate that Lys lowers the pKa of Tyr (Gerratana et al. 2001), which is normally between nine and 12. The measured pKa of Tyr in E. coli dTGDH (Gerratana et al. 2001) and E. coli GalE (Liu et al. 1997) is 6.4 and 6.1, respectively. The distance between the phenolic oxygen of Tyr150 and the amide nitrogen of Lys154 is 4.4 Å, a distance too far for hydrogen bonding, but within the range of electrostatic interactions to effectively lower the pKa of Tyr150.

The 4-keto intermediate acts as a springboard to other SDR reactions. In the case of GMD, the ketone functionality serves to acidify the proton at C5 and permits the dehydration from C6 to form the GDP-4-keto-5,6-ene intermediate. The presence of the "ene" intermediate has recently been detected in the homologous dTGDH reaction (Gross et al. 2000). For dehydration to occur, another active site base must be present to abstract a proton from the C5 position. Studies of E. coli GMD (Somoza et al. 2000) and dTGDH (Hegeman et al. 2001) showed that a glutamic acid might fulfill this requirement. The corresponding Glu128 side chain in GMD is within 3.6 Å of the C5 carbon of the mannose model, a position that would enable it to deprotonate C5. To complete the dehydration reaction, the C6 hydroxyl must be protonated by an active site acid. An Asp residue has been proposed based on structural analysis of dTGDH (Allard et al. 2002) and supported by mutagenesis experiments (Gross et al. 2001; Hegeman et al. 2001). The corresponding GMD residue, Ser127, is within 2.8 Å of the modeled position of the hexose O6 hydroxyl and may assume a similar role. Alternatively, Glu128 is within 2.6 Å, indicating the possibility of this side chain playing a dual role in the dehydration step, acting as both a general base and a general acid, as has been suggested in dTGDH and MUR1 (Hegeman et al. 2001; Mulichak et al. 2002). Whereas the dehydration mechanism described here is the step-wise water elimination mechanism as seen in D135N and D135A mutants of dTGDH, dehydration may also occur through a concerted mechanism as seen in wild-type dTGDH (Hegeman et al. 2002). Further kinetic studies would need to be completed to determine which mechanism of dehydration GMD actually uses.

The final step of the GMD reaction involves a hydride transfer from NADPH back to the hexose C6 position. The distance between the nicotinamide C4 atom and the C4 and C6 atoms of the hexose moiety (3.5 Å and 3.8 Å, respectively) indicates that only modest rotation of the hexose ring would be required to complete the hydride transfer. Interestingly, because NADPH is regenerated, the cofactor may remain bound through each catalytic cycle. To finalize the reduction step, an active site acid is required for proton addition to the C5 position of the hexose. Proposed residues to fulfill this role based on structural analysis of dTGDH include the catalytic Tyr, Glu, or Asp (Allard et al. 2002). Of the corresponding residues in GMD, Tyr150 and Ser127 (aligning with Asp) are >4.4 Å to the C5 position of the hexose model. Glu128 is 3.6 Å away but would move even further with the rotation of the hexose ring toward the nicotinamide ring. However, Thr126 of the catalytic triad and Asn179 are positioned such that they may be able to fulfill the role as the general acid to complete the reaction.

Structural comparisons
The secondary structural elements between P. aeruginosa GMD and MUR1 superimpose well with a root mean square deviation (RMSD) of 1.2 Å over C atoms. The secondary structural elements between P. aeruginosa GMD and E. coli GMD do not superimpose as well (RMSD, 3 Å) because E. coli GMD has no substrate or cofactor bound in the active site. The main difference between the three is an area of disorder present in MUR1 residues 76–81 and E. coli GMD residues 35–55, which corresponds to a region that is highly variable in size and sequence among GMDs (Fig. 5). This stretch in P. aeruginosa GMD is shorter and well ordered, forming a short helix (2) between 2 and 3 of the Rossmann fold. Immediately preceding this region is a Gly-XX-ArgArg sequence that is conserved among all GMDs sequenced thus far except for P. aeruginosa GMD. In contrast, P. aeruginosa GMD exhibits an arginine shift resulting in the sequence Gly-XXX-ArgArg. The two positively charged arginine residues mark the beginning of the RR loop that closes over the adenosyl phosphate end of the cofactor, are important in cofactor binding, and are involved in the tetramer interface for P. aeruginosa GMD and MUR1. The RR shift causes an interesting rearrangement of interactions. In MUR1 the first arginine of the sequence, Arg60, adopts a parallel stacking arrangement with Arg60* of the neighboring monomer, simultaneously packing against the adenine ring and coordinating the 2'-phosphate. In GMD the second arginine of the sequence Arg36 also adopts a parallel stacking arrangement with Arg36* of the neighboring monomer. However, because of the shift by two residues, the side chain is oriented almost perpendicular to the adenine ring (Fig. 6). Despite the rearrangement in this region, Arg36 in P. aeruginosa GMD still maintains the electrostatic interactions to coordinate the 2'-phosphate. Based on the role that the cofactor plays in the tetramer interface of P. aeruginosa GMD and MUR1, cofactor binding might also be expected to assist in ordering part of the RR loop in E. coli GMD and may be essential for tetramer formation.

View larger version (37K): [in this window] [in a new window]   Figure 6. Stereoview of the superposition of the RR loops from GMD (highlighted in color) and MUR1 (blue). GMD side chains are labeled in green, MUR1 side chains are labeled in blue, and asterisks denote side chains from the opposite monomer.

One of the intriguing features about P. aeruginosa GMD is its oligomeric state, as it deviates from the canonical homodimeric structures seen in most other related enzymes of the NDP-sugar modifying subfamily of the SDRs. The structures of all other members of this subfamily, including E. coli GMD, have been observed as dimers, with the exception of the previously mentioned tetrameric tyvelose epimerase (Koropatkin et al. 2003) and ADP-L-glycero-D-mannoheptose-6-epimerase, which is a pentamer (Deacon et al. 2000). P. aeruginosa GMD and A. thaliana MUR1 can be seen as a dimer of canonical SDR dimers, which then generates a new set of subunit interactions. An important subsequent question is whether a significant number of tetramer interactions between GMD and MUR1 are conserved. As previously mentioned, the RR loop of residues Arg35–Arg43, which is so intimately involved in the tetramer interface and the cofactor binding sites, makes protein–protein interactions as well as protein–cofactor interactions to the neighboring monomer. The Arg35 to Ser85* and Ser37 to Arg43* hydrogen bonds are conserved in GMD and MUR1. Furthermore, these residues are highly conserved among the GMD sequences. Also, within the RR loop is hydrogen bonding between Ser38 and Arg43*. Although the residue corresponding to Ser38 is an Asn in MUR1, the interaction is conserved. The sequences of several GMDs reveal that in most cases, a Ser is present in this position. Away from the cofactor binding site overlap, Asp62 hydrogen bonds to the amide nitrogen atoms of Val95* and Thr96*. Asp62 is highly conserved among GMDs. One of the more interesting interactions involves residue Arg68, which is moderately conserved across several GMD sequences. This residue is involved in hydrogen bonding to the main-chain carbonyl oxygen of Asn92*, an interaction also seen in MUR1. The same Arg in GMD and MUR1 extends toward the diagonally related monomer to hydrogen bond to Glu110 and Arg113, both highly conserved residues among GMD sequences.

In summary, the GMD MUR1 isoform from A. thaliana was shown to be a tetramer, whereas the first GMD structure to be determined, E. coli apo-GMD, was observed as a dimer. This raised the question as to whether or not prokaryotic and eukaryotic GMDs differed in oligomeric state. We have determined the structure of P. aeruginosa GMD with NADPH and GDP bound in the active site and found it to exist as a tetramer. The tetramer arises from the dimerization of the canonical dimer seen for most members of the SDR superfamily, but in a manner in which the cofactor binding sites closely interact across the new interface. The residues involved in the tetramer interactions are well conserved between the prokaryotic GMD and the eukaryotic MUR1. Moreover, a high degree of sequence conservation among the residues within the tetramer interface is also observed across a broad range of GMDs. These observations indicate that the tetramer may be a more common oligomeric state for GMDs than previously thought.

	Materials and methods

TOP Abstract Introduction Results and Discussion Materials and methods References

 
Expression and purification of GMD
The gmd gene, originally isolated by Currie et al. (1995), was amplified by PCR while incorporating a BamHI over the natural start codon and a HindIII site over the natural stop codon. The new PCR fragment was ligated into the pQE30 vector (Qiagen), placing a 6x-His tag at the N terminus. A glycerol stock of E. coli M15 cells harboring the recombinant plasmid was used to inoculate a starter culture of 50 mL Luria Bertani (LB) broth. After cultivation for 16 h at 37°C, the starter culture was transferred to 1 L LB broth. Once an OD₆₀₀ of 0.5 to 0.6 was reached, the cells were induced with 0.25 mM IPTG and grown for an additional 16 h at room temperature before harvesting by centrifugation. Cells were resuspended in lysis buffer (50 mM HEPES, 300 mM NaCl, 5 mM imidazole at pH 8.0) and lysed by sonication. The lysate was cleared by centrifugation at 10,000g for 20 min and loaded onto a column of 4 mL Ni-NTA resin (Qiagen). The column was washed with wash buffer (50 mM HEPES, 300 mM NaCl, 20 mM imidazole at pH 7.5), and protein was eluted with elution buffer (50 mM HEPES, 300 mM NaCl, 200 mM imidazole at pH 7.5). Further purification was carried out by FPLC using a HiTrap Q anion exchange column (Amersham Pharmacia Biotech) equilibrated with Buffer A (20 mM Tris at pH 8.5). Protein was eluted over a 40 mL salt gradient by using Buffer B (20 mM Tris at pH 8.5, 1 M NaCl). Fractions were analyzed by SDS-PAGE. Those shown to be homogenous were pooled and concentrated to 10 mg/mL.

Analysis of substrate conversion by capillary electrophoresis
The GMD assays were performed in 20 mM Tris-HCl at 37°C (pH 8.0), 50 mM MgCl₂, 10 mM GDP-D-mannose, and 10 mM NADP⁺ or NAD⁺, with a total reaction volume of 50 µL. The reaction was initiated by the addition of 1 to 10 µg of purified protein. At time periods between 10 and 30 min, samples were quenched by boiling for 3 min. Capillary electrophoresis (CE) on the reaction mixtures was performed by using a P/ACE MDQ System (Beckman Instruments) with UV detection. A 75 µm x 57 cm bare silica capillary was used with the UV detector mounted at 50 cm of the capillary. The CE analysis was run in 25 mM sodium tetraborate (pH 9.5), and sample was introduced by pressure injection for 8 sec. The separation was performed at 22 kV, and the sugar-nucleotide substrate and product were detected at 254 nm.

Crystallization
Purified GMD was used to set up crystallization screens (Crystal Screen 1 and 2 from Hampton Research) by using the hanging drop vapor diffusion method. Bipyramidal crystals were grown in a drop containing equal parts protein and well solution (50% 2-methyl-2,4-pentanediol, 100 mM Tris at pH 8.5, 200 mM NH₄H₂PO₄) and equilibrated against a reservoir of well solution. The resulting crystals (0.4 x 0.4 x 0.3 mm) belong to the trigonal space group P3₂21 (a = b = 125.7 Å, c = 220.0 Å, = = 90°, = 120°) with four molecules in the asymmetric unit. Prior to data collection, crystals were looped directly from the drop and flash-frozen in liquid propane.

Data collection and processing
X-ray diffraction data were collected to 2.2 Å on a MAR CCD detector at the Advanced Photon Source beamline 5-ID (DND), Argonne National Laboratory. During data collection the crystal was held at 100 K in a cryostream, and radiation was used at a 1.0 Å wavelength. The data were processed by using HKL, version 1.6, software (Otwinowski and Minor 1997). Data collection statistics are presented in Table 1.

	Acknowledgments

 
This work is supported by the Michigan Life Science Corridor grant to the Center for Structural Biology (R.M.G.) and a Canadian Cystic Fibrosis Foundation grant (J.S.L.). J.S.L. is a Canada Research Chair in Cystic Fibrosis and Microbial Glycobiology; the purchase of the CE equipment was funded by a Canadian Institute of Health Research equipment grant (no. MMA-41558, J.S.L.). Portions of this work were performed at the DuPont-Northwestern-Dow Collaborative Access Team (DND-CAT) Synchrotron Research Center located at Sector 5 of the Advanced Photon Source (APS). DND-CAT is supported by E.I. DuPont de Nemours & Co., Dow Chemical Company, the NSF grant DMR-9304725, and a grant from the State of Illinois (IBHE HECA NWU 96). We thank the staff of the DND-CAT beamline for their assistance.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

	References

TOP Abstract Introduction Results and Discussion Materials and methods References

 
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