The bacterial enzyme UDP-N-acetylglucosamine enolpyruvyl transferase catalyzes the first committed step of peptidoglycan biosynthesis, i.e., transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetylglucosamine. We have overexpressed the enzyme from Haemophilus influenzae in Escherichia coli and crystallized it in the apo-form, as well as in a complex with UDP-N-acetylglucosamine and fosfomycin using ammonium sulfate as the precipitant. X-ray diffraction data from a crystal of the apo-form were collected to 2.8 angstrom resolution at 293 K. The crystal quality was improved by co-crystallization with UDP-N-acetylglucosamine and fosfomycin. X-ray data to 2.2 angstrom have been collected at 100 K from a flash-frozen crystal of the complex. The complex crystals belong to the orthorhombic space group 1222 (or 12,2121) with unit-cell parameters of a = 63.7, b = 124.5, and c = 126.3 A. Assuming a monomer of the recombinant enzyme in the crystallographic asymmetric unit, the calculated Matthews parameter (V-M) is 2.71 angstrom(3) Da(-1) and solvent content is 54.6%.

The RecR protein plays a key role in the RecFOR pathway of recombination, which is necessary for the repair of ssDNA gaps. RecR from Deinococcus radiodurans has been overexpressed in Escherichia coli and crystallized at 297 K using polyethylene glycol 1000 as a precipitant. X-ray diffraction data to 2.90 A resolution have been collected at 100 K using Cu Kalpha X-rays from a mercury-soaked crystal. The crystal belongs to space group C222(1), with unit-cell parameters a = 106.96, b = 122.25, c = 156.01 A. The asymmetric unit contains four monomers of RecR, with a crystal volume per protein weight (V(M)) of 2.57 A(3) Da(-1) and a solvent content of 51.0%.

1,4-β-d-Xylan is the major component of plant cell-wall hemicelluloses. β-d-Xylosidases are involved in the breakdown of xylans into xylose and belong to families 3, 39, 43, 52, and 54 of glycoside hydrolases. Here, we report the first crystal structure of a member of family 39 glycoside hydrolase, i.e. β-d-xylosidase from Thermoanaerobacterium saccharolyticum strain B6A-RI. This study also represents the first structure of any β-xylosidase of the above five glycoside hydrolase families. Each monomer of T. saccharolyticum β-xylosidase comprises three distinct domains; a catalytic domain of the canonical (β/α)8-barrel fold, a β-sandwich domain, and a small α-helical domain. We have determined the structure in two forms: d-xylose-bound enzyme and a covalent 2-deoxy-2-fluoro-α-d-xylosyl-enzyme intermediate complex, thus providing two snapshots in the reaction pathway. This study provides structural evidence for the proposed double displacement mechanism that involves a covalent intermediate. Furthermore, it reveals possible functional roles for His228 as the auxiliary acid/base and Glu323 as a key residue in substrate recognition.

RecR, together with RecF and RecO, facilitates RecA loading in the RecF pathway of homologous recombinational DNA repair in procaryotes . The human Rad52 protein is a functional counterpart of RecFOR. We present here the crystal structure of RecR from Deinococcus radiodurans (DR RecR). A monomer of DR RecR has a two‐domain structure: the N‐terminal domain with a helix–hairpin–helix (HhH) motif and the C‐terminal domain with a Cys4 zinc‐finger motif, a Toprim domain and a Walker B motif. Four such monomers form a ring‐shaped tetramer of 222 symmetry with a central hole of 30−35 Å diameter. In the crystal, two tetramers are concatenated, implying that the RecR tetramer is capable of opening and closing. We also show that DR RecR binds to both dsDNA and ssDNA, and that its HhH motif is essential for DNA binding.

Acetohydroxy acid isomeroreductase (AHIR) is a key enzyme in the biosynthesis of branched-chain amino acids. We have determined the first crystal structure of a class I AHIR from Pseudomonas aeruginosa at 2.0Å resolution. Its dodecameric architecture of 23 point group symmetry is assembled of six dimeric units and dimerization is essential for the formation of the active site. The dimeric unit of P.aeruginosa AHIR partially superimposes with a three-domain monomer of spinach AHIR, a class II enzyme. This demonstrates that the so-called plant-specific insert in the middle of spinach AHIR is structurally and functionally equivalent to the C-terminal α-helical domain of P.aeruginosa AHIR, and the C-terminal α-helical domain was duplicated during evolution from the shorter, class I AHIRs to the longer, class II AHIRs. The dimeric unit of P.aeruginosa AHIR possesses a deep figure-of-eight knot, essentially identical with that in the spinach AHIR monomer. Thus, our work lowers the likelihood of the previous proposal that “domain duplication followed by exchange of a secondary structure element can be a source of such a knot in the protein structure” being correct.

tRNA(m1G37)methyltransferase (TrmD) catalyzes the transfer of a methyl group from S‐adenosyl‐L‐ methionine (AdoMet) to G37 within a subset of bacterial tRNA species, which have a G residue at the 36th position. The modified guanosine is adjacent to and 3′ of the anticodon and is essential for the maintenance of the correct reading frame during translation. Here we report four crystal structures of TrmD from Haemophilus influenzae, as binary complexes with either AdoMet or S‐adenosyl‐L‐homocysteine (AdoHcy), as a ternary complex with AdoHcy and phosphate, and as an apo form. This first structure of TrmD indicates that it functions as a dimer. It also suggests the binding mode of G36G37 in the active site of TrmD and the catalytic mechanism. The N‐terminal domain has a trefoil knot, in which AdoMet or AdoHcy is bound in a novel, bent conformation. The C‐terminal domain shows structural similarity to trp repressor. We propose a plausible model for the TrmD2–tRNA2 complex, which provides insights into recognition of the general tRNA structure by TrmD.

Lipid A is the hydrophobic anchor of lipopolysaccharide in Gram-negative bacteria and is required for growth of most Gram-negative bacteria.1, 2 It is also necessary for maintaining the integrity of the outer membrane as a barrier to toxic chemicals.3, 4 Therefore, the study of the enzymes involved in lipid A biosynthesis would be useful for the development of new antibacterial drugs against Gram-negative bacteria.5 UDP-N-acetylglucosamine acyltransferase (LpxA) is the first enzyme of the lipid A biosynthetic pathway. It catalyzes the transfer of an R-3-hydroxyacyl chain from R-3-hydroxy-acyl carrier protein (ACP) to UDP-N-acetylglucosamine (UDP-GlcNAc) at the glucosamine 3-OH position. Among nine enzymes of lipid A biosynthetic pathway, information on the 3D structure is available on LpxA from Escherichia coli only.6 It is a trimer composed of three identical subunits of 262 residues and contains a left-handed parallel β-helix motif. However, the crystal structure of E. coli LpxA determined at 2.6-Å resolution did not contain any bound ligand and provided little information on the active site. The interaction site of LpxA involved in binding ACP is also unknown. Therefore, further structural data on LpxA will be valuable for a better understanding of the active site and structure-based inhibitor design. Here, we present the crystal structure of LpxA from Heliobacter pylori refined using 2.1-Å data. The sequence identity between H. pylori LpxA (270 residues, 29,855 Da) and that from E. coli is 39.3% over the entire polypeptide chain. Thanks to higher resolution, we could assign solvent molecules as well as bound ions. Further, an extra electron density is present in the putative active site and we tentatively interpret this unknown ligand as a detergent molecule, which seems to mimic the acyl chain of the substrate or the product. On the basis of this observation, together with the location of strictly conserved residues and the highly positively charged surface of the C-terminal helical domain, we propose a model for the complex between LpxA and ACP. Overexpression of H. pylori LpxA as a fusion with a C-terminal eight-residue tag, its crystallization, and X-ray data collection have been reported elsewhere.7 The structure was solved by the molecular replacement method using the 2.6-Å structure of E. coli LpxA6 [Protein Data Bank (PDB) ID 1LXA] as a search model. The model of H. pylori LpxA (PDB ID 1J2Z) has been refined to crystallographic Rwork and Rfree values of 22.2 and 26.4%, respectively, for reflections with F > 2σ in the resolution range 20–2.1 Å. It consists of 2001 nonhydrogen protein atoms from 259 amino acid residues (residues 2–260) in a monomer, 1 tartrate ion, 2 sulfate ions, 1 detergent molecule (1-s-octyl-β-D-thioglucoside), and 148 water molecules in the asymmetrical unit. One-hundred ninety residues (85.6%) of 222 nonglycine and nonproline residues are in the most favored regions of the Ramachandran plot and 32 residues (14.4%) in the additionally allowed regions. Refinement statistics are shown in Table I. Compared with most other LpxAs, H. pylori LpxA is longer at its C-terminus by about 10 residues. This C-terminal extension (residues 261–270) and eight residues from the C-terminal tag are not visible in the electron density map, presumably because they are disordered in the crystal. H. pylori LpxA is a homo-trimer in its quaternary structure [Fig. 1(A)] and its subunit possesses a highly similar fold as that of E. coli LpxA.6 The root mean square (RMS) difference between H. pylori and E. coli enzymes is 1.22 Å for 229 Cα atom pairs. Each subunit can be divided into two distinct domains. The N-terminal domain (residues 2–186) is folded into the left-handed parallel β-helix motif (LβH), which comprises 28 β-strands. The first 27 β-strands are wound in 9 helical turns and the last β-strand is only one third of a helical turn [Fig. 1(B)]. The fourth turn has a 12-residue insertion between strands β11 and β12, and the fifth turn has another 7-residue insertion between β14 and β15, respectively. Theses insertions cover the strictly conserved residues (His118, His121, His140), which could possibly play important catalytic roles. Other turns except the ninth turn comprise exactly 18 amino acid residues in three β-strands. Each turn of the β-helix is approximately triangular and three β-helix motifs pack nicely into a trimer. The trimer formation is critical for the catalytic function of LpxA, as discussed below. Structure of H. pylori UDP-N-acetylglucosamine acyltransferase. A: Trimeric architecture. Side-chains of the strictly conserved residues Glu90, His118, His121, His140, Gln157, Asn194, and Arg199 are blue. B: Monomer fold. A molecule of 1-s-octyl-β-D-thioglucoside is also shown. This view is obtained by a slight rotation of the green subunit in A around a vertical axis. C: Stereo view of the proposed model of the complex between H. pylori LpxA and E. coli ACP.8 This is the same view as in A. The molecular surface is colored according to the electrostatic potential: blue, +10 kT; white, 0 kT; red, −10 kT. Butyrylated 4′-phosphopantetheine group (pink) is covalently attached to Ser36 in E. coli ACP. Taking into account all the available information, we also built UDP-GlcNAc (red, yellow, and white) into the model just for the sake of completeness; however, we emphasize that UDP-GlcNAc was not included in the crystallization medium. The C-terminal helical domain (residues 187–260) contains four α-helices. Three C-terminal domains in a trimer do not contact each other and they provide a highly positively charged surface at the bottom side of the trimeric enzyme [Fig. 1(C)]. Because H. pylori and E. coli ACPs are highly acidic with calculated pI values of 3.85 and 3.98, respectively, we suggest that this positively charged surface at the bottom of the trimer is likely to be the binding site for ACP. In a crude docking attempt, three molecules of E. coli ACP8 can be positioned nicely into three identical pockets, each of which is formed between the two subunits. The proposed binding site of ACP is further supported by the distribution of strictly conserved residues. Two strictly conserved residues, Asn194 and Arg199, are located at N-terminal and C-terminal ends of the helix α1 in the C-terminal domain, respectively. Their side-chains point toward the proposed ACP binding site, which is adjacent to the putative catalytic site, as defined by other strictly conserved residues (discussed below). In the proposed binding model, Arg199 is in proximity of a highly negatively charged surface patch of ACP. The residues of the C-terminal domain on the other side of the proposed ACP binding pocket are not highly conserved and it appears that ACP interacts mainly through the face of the binding pocket lined with strictly conserved Asn194 and Arg199. Among 26 LpxA sequences, 11 residues are strictly conserved: 9 residues (Gly53, Gly83, Glu90, Gly108, His118, His121, Gly139, His140, Gln157) in the N-terminal β-helix domain and 2 (Asn194, Arg199) in the C-terminal helical domain. Four conserved glycine residues are most likely to play a structural role. The role of the strictly conserved Glu90 is unclear from the structure; it is located at the subunit interface and is a little separated from residues His118, His121, His140, and Gln157, which are clustered underneath the insertion loops of the β-helix motif. Three histidine residues, His118(His122), His140(His144), and His156(His160), were implicated for the substrate binding through chemical modification and site-directed mutagenesis of E. coli LpxA.9 The corresponding residues of E. coli LpxA are given in parentheses. In addition, Lys72(Lys76) and Arg199*(Arg204*) have also been suggested to play some roles in substrate binding. An asterisk after the residue number denotes that the residue comes from a neighboring subunit. Lys72(Lys76) and His156(His160) are less strictly conserved and are likely to play a minor role in substrate binding. His121(His125) and Gln157(Gln161) are likely to play an important role in either catalysis or substrate binding. His121(His125) was proposed to act as a general base to increase the nucleophilicity of the glucosamine 3-OH of UDP-GlcNAc during its attack on the thioester carbonyl of the acyl-ACP donor.9 As mentioned above, Asn194*(Asn199*) and Arg199*(Arg*204) from the C-terminal α-helical domain of the neighboring subunit are likely to be important in recognizing the holo-ACP, in particular its conserved features such as 4′-phosphopantetheine group and a negatively charged surface patch. An extra electron density for an unknown ligand is present in the putative active site of H. pylori LpxA. We tentatively interpret it as a molecule of the detergent 1-s-octyl-β-D-thioglucoside, which was added to the hanging drop and was found to be necessary for crystallization.7 The strictly conserved residues His118, His121, His140, and Gln157 point toward the extra electron density and they clearly define the location of the catalytic site. The last carbon atom at the end of the octyl group in the detergent is ≈10 Å from the Cα atom of Gly169*. When the octyl chain is extended by six more carbon atoms, the distance becomes ≈2.5 Å. Gly173 of E. coli LpxA and Met169 of Pseudomonas aeruginosa LpxA, corresponding to Gly169 of H. pylori LpxA, were found to determine the specificity for the acyl chain length.10 E. coli LpxA is selective for the 14-carbon myristoyl chain, whereas P. aeruginosa LpxA is selective to the 10-carbon decanoyl chain.10 The side-chain oxygen atom of Gln157 lies ≈4 Å from the C3 atom of the octyl group. This indicates that Gln157 may be important in recognizing the hydroxyl group of the R-3-hydroxymyristoyl chain. And, H. pylori LpxA is most likely to prefer the myristoyl chain like E. coli LpxA. The authors thank Prof. N. Sakabe and his staff for assistance during data collection at beamline BL-18B of Photon Factory, Japan. This work was supported by the Center for Functional Analysis of Human Genome (21st Century Frontier Program) of the Korea Ministry of Science and Technology. B.I.L. is supported by the BK21 Fellowship.

Lipid A, a constituent of lipopolysaccharides, is essential for the growth and virulence of most Gram-negative bacteria. This makes its biosynthetic enzymes potential targets for development of new antibacterial agents. The first step of lipid A biosynthesis is catalyzed by the enzyme UDP-N-acetylglucosamine acyltransferase (LpxA). LpxA from the pathogenic bacterium Helicobacter pylori has been overexpressed in Escherichia coli and crystallized at 297 K using ammonium sulfate and sodium/potassium tartrate as precipitants in the presence of a detergent. Diffraction data to 2.1 A resolution have been collected from a native crystal. The crystal belongs to space group P6(3)22, with unit-cell parameters a = b = 90.69, c = 148.20 A. The asymmetric unit contains one subunit of LpxA, with a crystal volume per protein mass (V(M)) of 2.87 A(3) Da(-1) and a solvent content of 57.1%.

beta-Xylosidases are involved in the breakdown of xylans into xylose and belong to either family 39 or 43 of the glycosyl hydrolases. At present, no structural information is available for any member of these families. beta-Xylosidase from the thermophilic anaerobe Thermoanaerobacterium saccharolyticum, a member of glycosyl hydrolase family 39, has been crystallized at 296 K using the hanging-drop vapour-diffusion method. The crystal diffracts to 2.4 A resolution with synchrotron X-rays and belongs to space group P4(1)2(1)2 (or P4(3)2(1)2), with unit-cell parameters a = b = 92.75, c = 241.37 A. The asymmetric unit contains two monomers of the recombinant enzyme, giving a corresponding V(M) of 2.21 A(3)Da(-1) and a solvent content of 44.3%.

Acetohydroxy acid isomeroreductase (AHIR) is involved in the biosynthetic pathway of branched-chain amino acids in microorganisms and plants. AHIR from Pseudomonas aeruginosa has been overexpressed in Escherichia coli and crystallized at 297 K using potassium/sodium tartrate as a precipitant. X-ray diffraction data have been collected to 2.0 A resolution at 100 K using synchrotron radiation. The crystals belong to the cubic space group P2(1)3, with unit-cell parameters a = b = c = 184.38 A, alpha = beta = gamma = 90 degrees. Six monomers are present in the asymmetric unit, giving a V(M) of 2.34 A(3) Da(-1) and a solvent content of 47.4%.

Aspartate 1-decarboxylase (PanD) catalyzes the alpha-decarboxylation of L-aspartate in the major route of beta-alanine production for pantothenate biosynthesis in bacteria. Pantothenate is synthesized in microorganisms, plants and fungi but not in animals and thus the enzymes of its biosynthetic pathway are potential targets for developing agents against these organisms. PanD from the pathogenic bacterium Helicobacter pylori has been overexpressed in Escherichia coli and crystallized using sodium formate as a precipitant. Crystals diffracted to better than 1.5 A Bragg spacing upon exposure to synchrotron X-rays. Diffraction data to 1.55 A have been collected from a crystal grown in the presence of the substrate analogue isoasparagine. The crystal belongs to the tetragonal space group I422, with unit-cell parameters a = b = 81.83, c = 93.78 A. The asymmetric unit contains one subunit of PanD, with a corresponding crystal volume per protein mass (V(M)) of 2.85 A(3) Da(-1) and a solvent content of 56.8%.

The MJ0490 gene, one of the only two genes of Methanococcus jannaschii showing sequence similarity to the lactate/malate family of dehydrogenases, was classified initially as coding for a putative l-lactate dehydrogenase (LDH). It has been re-classified as a malate dehydrogenase (MDH) gene, because it shows significant sequence similarity to MT0188, MDH II from Methanobacterium thermoautotrophicum strain DeltaH. The three-dimensional structure of its gene product has been determined in two crystal forms: a "dimeric" structure in the orthorhombic crystal at 1.9 A resolution and a "tetrameric" structure in the tetragonal crystal at 2.8 A. These structures share a similar subunit fold with other LDHs and MDHs. The tetrameric structure resembles typical tetrameric LDHs. The dimeric structure is equivalent to the P-dimer of tetrameric LDHs, unlike dimeric MDHs, which correspond to the Q-dimer. The structure reveals that the cofactor NADP(H) is bound at the active site, despite the fact that it was not intentionally added during protein purification and crystallization. The preference of NADP(H) over NAD(H) has been supported by activity assays. The cofactor preference is explained by the presence of a glycine residue in the cofactor binding pocket (Gly33), which replaces a conserved aspartate (or glutamate) residue in other NAD-dependent LDHs or MDHs. Preference for NADP(H) is contributed by hydrogen bonds between the oxygen atoms of the monophosphate group and the ribose sugar of adenosine in NADP(H) and the side-chains of Ser9, Arg34, His36, and Ser37. The MDH activity of MJ0490 is made possible by Arg86, which is conserved in MDHs but not in LDHs. The enzymatic assay showed that the MJ0490 protein possesses the fructose-1,6-bisphosphate-activated LDH activity (reduction). Thus the MJ0490 gene product appears to be a novel member of the lactate/malate dehydrogenase family, displaying an LDH scaffold and exhibiting a relaxed substrate and cofactor specificities in NADP(H) and NAD(H)-dependent malate and lactate dehydrogenase reactions.

A 110-residue protein encoded by the TM1442 gene of Thermotoga maritima shows amino-acid sequence similarity to Bacillus subtilis anti-anti-sigma factors RsbV and SpoIIAA. It has been overexpressed in Escherichia coli and the recombinant protein exists primarily as both a monomer and a dimer in solution. The dimeric form has been crystallized using polyethylene glycol (PEG) 8000 as a precipitant. Native X-ray diffraction data have been collected at 100 K to 2.0 A resolution. The crystals are monoclinic, belonging to the space group P2(1), with unit-cell parameters a = 31.54 (13), b = 116.83 (37), c = 31.39 (7) A, alpha = 90, beta = 119.84 (9), gamma = 90 degrees. The asymmetric unit contains two monomers of the recombinant polypeptide, with a corresponding V(M) of 2.24 A(3) Da(-1) and a solvent content of 45.0%.

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) from Saccharomyces cerevisiae is essential for cell viability. It has been overexpressed in Escherichia coli and has been crystallized at 296 K using polyethylene glycol (PEG) 1500 as a precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 59.48, b = 138.54, c = 157.91 A, alpha = beta = gamma = 90 degrees. Two molecules of trimeric dUTPase from S. cerevisiae are present in the asymmetric unit, giving a crystal volume per protein mass (V(M)) of 3.36 A(3) Da(-1) and a solvent content of 63%. The diffraction limit of the crystals could be significantly extended by the crystal-annealing procedure. A set of native data extending to 2.7 A resolution has been collected at 100 K using synchrotron X-rays.

L(+)-Lactate dehydrogenase (LDH) is a key enzyme in anaerobic metabolism which converts pyruvate to lactate. LDH from the hyperthermophilic archaebacterium Methanococcus jannaschii has been overexpressed in Escherichia coli and crystallized in two crystal forms at 297 K using 2-methyl-2,4-pentanediol as precipitant. Type I crystals grew rapidly and diffracted to at least 2.8 A Bragg spacing upon exposure to Cu Kalpha X-rays. X-ray diffraction data to 2.9 A have been collected from a native crystal. The type I crystal is tetragonal, belonging to the space group P4(2)2(1)2, with unit-cell parameters a = b = 99.74, c = 170.00 A. The asymmetric unit contains two LDH subunits, with a corresponding crystal volume per protein mass (V(m)) of 3.05 A(3) Da(-1) and a solvent content of 59.7%. Type II crystals, which grew more slowly, diffracted to at least 1.8 A Bragg spacing upon exposure to Cu Kalpha X-rays. X-ray diffraction data to 1.9 A have been collected from a native crystal. The type II crystal is orthorhombic, belonging to the space group P2(1)2(1)2, with unit-cell parameters a = 47.65, b = 125.10, c = 58.08 A. The asymmetric unit contains a single LDH subunit, with a corresponding crystal volume per protein mass (V(m)) of 2.50 A(3) Da(-1) and a solvent content of 50.8%. Therefore, the type II crystal is more suitable for high-resolution structure determination than the type I crystal.