The Structure of the Neisserial Lipooligosaccharide Phosphoethanolamine Transferase A (LptA) Required for Resistance to Polymyxin

Gram-negative bacteria possess an outer membrane envelope consisting of an outer leaflet of lipopolysaccharides, also called endotoxins, which protect the pathogen from antimicrobial peptides and have multifaceted roles in virulence. Lipopolysaccharide consists of a glycan moiety attached to lipid A, embedded in the outer membrane. Modification of the lipid A headgroups by phosphoethanolamine (PEA) or 4-amino-arabinose residues increases resistance to the cationic cyclic polypeptide antibiotic, polymyxin. Lipid A PEA transferases are members of the YhjW/YjdB/YijP superfamily and usually consist of a transmembrane domain anchoring the enzyme to the periplasmic face of the cytoplasmic membrane attached to a soluble catalytic domain. The crystal structure of the soluble domain of the protein of the lipid A PEA transferase from Neisseria meningitidis has been determined crystallographically and refined to 1.4 Å resolution. The structure reveals a core hydrolase fold similar to that of alkaline phosphatase. Loop regions in the structure differ, presumably to enable interaction with the membrane-localized substrates and to provide substrate specificity. A phosphorylated form of the putative nucleophile, Thr280, is observed. Metal ions present in the active site are coordinated to Thr280 and to residues conserved among the family of transferases. The structure reveals the protein components needed for the transferase chemistry; however, substrate-binding regions are not evident and are likely to reside in the transmembrane domain of the protein.     

Ring-opening mechanism revealed by crystal structures of NagB and its ES intermediate complex

Glucosamine 6-phosphate deaminase (NagB) catalyzes the conversion of d-glucosamine 6-phosphate (GlcN6P) to d-fructose 6-phosphate and ammonia. This reaction is the final step of N-acetylglucosamine utilization and decides its metabolic fate. The enzyme from Streptococcus mutans belongs to the monomeric subfamily of NagB. The crystal structure of the native SmuNagB (NagB from S. mutans) presented here, compared with the structures of its homologs BsuNagB (NagB from Bacillus subtilis) and EcoNagB (NagB from E. coli), implies a conformational change of the ‘lid’ motif in the activation of the monomeric NagB enzyme. We have also captured the enzyme-substrate intermediate complex of the NagB family at low pH, where a remarkable loss of the catalytic activity of SmuNagB was detected. The enzyme-substrate intermediate presents the initial step of the GlcN6P deaminase reaction. The structural evidence (1) supports the α-anomer of GlcN6P as the specific natural substrate of NagB; (2) displays the substrate-binding pocket at the active site; and (3) together with the site-directed mutagenesis studies, demonstrates the ring-opening mechanism of an Asn-His-Glu triad that performs the proton transfer from O1 to O5 to open the sugar ring.     

Crystal Structure of Metal-Dependent Allantoinase from Escherichia coli

Allantoinase acts as a key enzyme for the biogenesis and degradation of ureides by catalyzing the conversion of (S)-allantoin into allantoate, the final step in the ureide pathway. Despite limited sequence similarity, biochemical studies of the enzyme suggested that allantoinase belongs to the amidohydrolase family. In this study, the crystal structure of allantoinase from Escherichia coli was determined at 2.1 Å resolution. The enzyme consists of a homotetramer in which each monomer contains two domains: a pseudo-triosephosphate-isomerase barrel and a β-sheet. Analogous to other enzymes in the amidohydrolase family, allantoinase retains a binuclear metal center in the active site, embedded within the barrel fold. Structural analyses demonstrated that the metal ions in the active site ligate one hydroxide and six residues that are conserved among allantoinases from other organisms. Functional analyses showed that the presence of zinc in the metal center is essential for catalysis and enantioselectivity of substrate. Both the metal center and active site residues Asn94 and Ser317 play crucial roles in dictating enzyme activity. These structural and functional features are distinctively different from those of the metal-independent allantoinase, which was very recently identified.     

Structural and kinetic characterization of quinolinate phosphoribosyltransferase (hQPRTase) from homo sapiens

Human quinolinate phosphoribosyltransferase (EC 2.4.2.19) (hQPRTase) is a member of the type II phosphoribosyltransferase family involved in the catabolism of quinolinic acid (QA). It catalyses the formation of nicotinic acid mononucleotide from quinolinic acid, which involves a phosphoribosyl transfer reaction followed by decarboxylation. hQPRTase has been implicated in a number of neurological conditions and in order to study it further, we have carried out structural and kinetic studies on recombinant hQPRTase. The structure of the fully active enzyme overexpressed in Escherichia coli was solved using multiwavelength methods to a resolution of 2.0 A. hQPRTase has a alpha/beta barrel fold sharing a similar overall structure with the bacterial QPRTases. The active site of hQPRTase is located at an alpha/beta open sandwich structure that serves as a cup for the alpha/beta barrel of the adjacent subunit with a QA binding site consisting of three arginine residues (R102, R138 and R161) and two lysine residues (K139 and K171). Mutation of these residues affected substrate binding or abolished the enzymatic activity. The kinetics of the human enzyme are different to the bacterial enzymes studied, hQPRTase is inhibited competitively and non-competitively by one of its substrates, 5-phosphoribosylpyrophosphate (PRPP). The human enzyme adopts a hexameric arrangement, which places the active sites in close proximity to each other.     

Structure of the GcpE (IspG)-MEcPP complex from Thermus thermophilus

Isoprenoid precursor biosynthesis occurs through the mevalonate or the methylerythritol phosphate (MEP) pathway, used i.e., by humans and by many human pathogens, respectively. In the MEP pathway, 2-C-methyl-d-erythritol-2,4-cyclo-diphosphate (MEcPP) is converted to (E)-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate (HMBPP) by the iron-sulfur cluster enzyme HMBPP synthase (GcpE). The presented X-ray structure of the GcpE-MEcPP complex from Thermus thermophilus at 1.55 Å resolution provides valuable information about the catalytic mechanism and for rational inhibitor design. MEcPP binding inside the TIM-barrel funnel induces a 60° rotation of the [4Fe-4S] cluster containing domain onto the TIM-barrel entrance. The apical iron of the [4Fe-4S] cluster ligates with the C3 oxygen atom of MEcPP.     

Crystal structure of YihS in complex with D-mannose: structural annotation of Escherichia coli and Salmonella enterica yihS-encoded proteins to an aldose-ketose isomerase

The three-dimensional structure of a Salmonella enterica hypothetical protein YihS is significantly similar to that of N-acyl-d-glucosamine 2-epimerase (AGE) with respect to a common scaffold, an α₆/α₆-barrel, although the function of YihS remains to be clarified. To identify the function of YihS, Escherichia coli and S. enterica YihS proteins were overexpressed in E. coli, purified, and characterized. Both proteins were found to show no AGE activity but showed cofactor-independent aldose-ketose isomerase activity involved in the interconversion of monosaccharides, mannose, fructose, and glucose, or lyxose and xylulose. In order to clarify the structure/function relationship of YihS, we determined the crystal structure of S. enterica YihS mutant (H248A) in complex with a substrate (d-mannose) at 1.6 Å resolution. This enzyme-substrate complex structure is the first demonstration in the AGE structural family, and it enables us to identify active-site residues and postulate a reaction mechanism for YihS. The substrate, β-d-mannose, fits well in the active site and is specifically recognized by the enzyme. The substrate-binding site of YihS for the mannose C1 and O5 atoms is architecturally similar to those of mutarotases, suggesting that YihS adopts the pyranose ring-opening process by His383 and acidifies the C2 position, forming an aldehyde at the C1 position. In the isomerization step, His248 functions as a base catalyst responsible for transferring the proton from the C2 to C1 positions through a cis-enediol intermediate. On the other hand, in AGE, His248 is thought to abstract and re-adduct the proton at the C2 position of the substrate. These findings provide not only molecular insights into the YihS reaction mechanism but also useful information for the molecular design of novel carbohydrate-active enzymes with the common scaffold, α₆/α₆-barrel.     

Unexpected active-site flexibility in the structure of human neutrophil elastase in complex with a new dihydropyrimidone inhibitor

Human neutrophil elastase (HNE), a trypsin-type serine protease, is of pivotal importance in the onset and progression of chronic obstructive pulmonary disease (COPD). COPD encompasses a group of slowly progressive respiratory disorders and is a major medical problem and the fifth leading cause of death worldwide. HNE is a major target for the development of compounds that inhibit the progression of long-term lung function decline in COPD patients.Here, we present the three-dimensional structure of a potent dihydropyrimidone inhibitor (DHPI) non-covalently bound to HNE at a resolution of 2.0 Å. The inhibitor binds to the active site in a unique orientation addressing S1 and S2 subsites of the protease. To facilitate further analysis of this binding mode, we determined the structure of the uncomplexed enzyme at a resolution of 1.86 Å. Detailed comparisons of the HNE:DHPI complex with the uncomplexed HNE structure and published structures of other elastase:inhibitor complexes revealed that binding of DHPI leads to large conformational changes in residues located in the S2 subsite. The rearrangement of residues Asp95-Leu99B creates a deep, well-defined cavity, which is filled by the P2 moiety of the inhibitor molecule to almost perfect shape complementarity. The shape of the S2 subsite in complex with DHPI clearly differs from all other observed HNE structures. The observed structural flexibility of the S2 subsite is a key feature for the understanding of the binding mode of DHPIs in general and the development of new HNE selective inhibitors.     

Crystal structure of the parasite protease inhibitor chagasin in complex with a host target cysteine protease

Chagasin is a protein produced by Trypanosoma cruzi, the parasite that causes Chagas' disease. This small protein belongs to a recently defined family of cysteine protease inhibitors. Although resembling well-known inhibitors like the cystatins in size (110 amino acid residues) and function (they all inhibit papain-like (C1 family) proteases), it has a unique amino acid sequence and structure. We have crystallized and solved the structure of chagasin in complex with the host cysteine protease, cathepsin L, at 1.75 A resolution. An inhibitory wedge composed of three loops (L2, L4, and L6) forms a number of contacts responsible for high-affinity binding (K(i), 39 pM) to the enzyme. All three loops interact with the catalytic groove, with the central loop L2 inserted directly into the catalytic center. Loops L4 and L6 embrace the enzyme molecule from both sides and exhibit distinctly different patterns of protein-protein recognition. Comparison with a 1.7 A structure of uncomplexed chagasin, also determined in this study, demonstrates that a conformational change of the first binding loop (L4) allows extended binding to the non-primed substrate pockets of the enzyme active site cleft, thereby providing a substantial part of the inhibitory surface. The mode of chagasin binding is generally similar, albeit distinctly different in detail, when compared to those displayed by cystatins and the cysteine protease inhibitory p41 fragment of the invariant chain. The chagasin-cathepsin L complex structure provides details of how the parasite protein inhibits a host enzyme of possible importance in host defense. The high level of structural and functional similarity between cathepsin L and the T. cruzi enzyme cruzipain gives clues to how the cysteine protease activity of the parasite can be targeted. This information will aid in the development of synthetic inhibitors for use as potential drugs for the treatment of Chagas disease.     

Isolation of an N-acetyl-D-glucosamine specific lectin from the rhizomes of Arundo donax with antiproliferative activity

A lectin with antiproliferative activity towards human cancer cell lines and mitogenic towards human peripheral blood mononuclear cells was purified from the rhizomes of Arundo donax (Linn.) by affinity chromatography on N-acetyl-d-glucosamine linked to epoxy-activated sepharose-6B. The pure preparation apparently yielded a single band of approximately 15 kDa on SDS-PAGE, pH 8.3, under both reducing and non-reducing conditions. The molecular mass of native lectin was 32 kDa as determined by gel filtration chromatography. This showed the lectin to be a dimer, with subunits not held together by disulphide linkages. The A. donax lectin (ADL) agglutinated rabbit erythrocytes and the agglutination was inhibited by N-acetyl-d-glucosamine and its di- and trimer. The lectin was thermostable upto 55 degrees C and showed optimum activity in the range of pH 7.0-9.0 and comprised of 2.1% carbohydrate content.     

Crystal Structure of Mouse MD-1 with Endogenous Phospholipid Bound in Its Cavity

MD-1 is a glycoprotein that associates with a B-cell-specific RP105 protein and has a low sequence identity of 16% to MD-2 that associates with Toll-like receptor 4 and recognizes endotoxic lipopolysaccharide. MD-1 and RP105 are supposed to mediate lipopolysaccharide recognition; however, little is known about their structures and functions. Here, the crystal structure of mouse MD-1 is determined at 1.65 Å resolution. MD-1 has a hydrophobic cavity sandwiched by two β-sheets as is MD-2. The cavity is 25 Å long, 5 Å wide, and 10 Å deep: longer, narrower, and shallower than that of MD-2. No charged residues are located on the cavity entrance. MD-1 is primarily monomeric in solution but shows a dimeric assembly in the crystal lattices, with their cavity entrances facing each other. In the cavity, electron densities attributable to phosphatidylcholine are located. Together with the binding assay with tetra-acylated lipid IVa, MD-1 is shown to be a lipid-binding coreceptor.     

The Crystal Structure of the [NiFe] Hydrogenase from the Photosynthetic Bacterium Allochromatium vinosum: Characterization of the Oxidized Enzyme (Ni-A State)

The crystal structure of the membrane-associated [NiFe] hydrogenase from Allochromatium vinosum has been determined to 2.1 Å resolution. Electron paramagnetic resonance (EPR) and Fourier transform infrared spectroscopy on dissolved crystals showed that it is present in the Ni-A state (> 90%). The structure of the A. vinosum [NiFe] hydrogenase shows significant similarities with [NiFe] hydrogenase structures derived from Desulfovibrio species. The amino acid sequence identity is ∼ 50%. The bimetallic [NiFe] active site is located in the large subunit of the heterodimer and possesses three diatomic non-protein ligands coordinated to the Fe (two CN⁻ , one CO). Ni is bound to the protein backbone via four cysteine thiolates; two of them also bridge the two metals. One of the bridging cysteines (Cys64) exhibits a modified thiolate in part of the sample. A mono-oxo bridging ligand was assigned between the metal ions of the catalytic center. This is in contrast to a proposal for Desulfovibrio sp. hydrogenases that show a di-oxo species in this position for the Ni-A state. The additional metal site located in the large subunit appears to be a Mg²⁺ ion. Three iron-sulfur clusters were found in the small subunit that forms the electron transfer chain connecting the catalytic site with the molecular surface. The calculated anomalous Fourier map indicates a distorted proximal iron-sulfur cluster in part of the crystals. This altered proximal cluster is supposed to be paramagnetic and is exchange coupled to the Ni³⁺ ion and the medial [Fe₃S₄]⁺ cluster that are both EPR active (S = 1/2 species). This finding of a modified proximal cluster in the [NiFe] hydrogenase might explain the observation of split EPR signals that are occasionally detected in the oxidized state of membrane-bound [NiFe] hydrogenases as from A. vinosum.     

Crystal Structure of Bacillus cereusd-Alanyl Carrier Protein Ligase (DltA) in Complex with ATP

d-Alanylation of lipoteichoic acids modulates the surface charge and ligand binding of the Gram-positive cell wall. Disruption of the bacterial dlt operon involved in teichoic acid alanylation, as well as inhibition of the DltA (d-alanyl carrier protein ligase) protein, has been shown to render the bacterium more susceptible to conventional antibiotics and host defense responses. The DltA catalyzes the adenylation and thiolation reactions of d-alanine. This enzyme belongs to a superfamily of AMP-forming domains such as the ubiquitous acetyl-coenzyme A synthetase. We have determined the 1.9-Å-resolution crystal structure of a DltA protein from Bacillus cereus in complex with ATP. This structure sheds light on the geometry of the bound ATP. The invariant catalytic residue Lys492 appears to be mobile, suggesting a molecular mechanism of catalysis for this superfamily of enzymes. Specific roles are also revealed for two other invariant residues: the divalent cation-stabilizing Glu298 and the β-phosphate-interacting Arg397. Mutant proteins with a glutamine substitution at position 298 or 397 are inactive.     

Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with D-tagatose and D-fructose

Pseudomonas cichoriiid-tagatose 3-epimerase (P. cichoriid-TE) can efficiently catalyze the epimerization of not only d-tagatose to d-sorbose, but also d-fructose to d-psicose, and is used for the production of d-psicose from d-fructose. The crystal structures of P. cichoriid-TE alone and in complexes with d-tagatose and d-fructose were determined at resolutions of 1.79, 2.28, and 2.06 Å, respectively. A subunit of P. cichoriid-TE adopts a (β/α)₈ barrel structure, and a metal ion (Mn²⁺) found in the active site is coordinated by Glu152, Asp185, His211, and Glu246 at the end of the β-barrel. P. cichoriid-TE forms a stable dimer to give a favorable accessible surface for substrate binding on the front side of the dimer. The simulated omit map indicates that O2 and O3 of d-tagatose and/or d-fructose coordinate Mn²⁺, and that C3-O3 is located between carboxyl groups of Glu152 and Glu246, supporting the previously proposed mechanism of deprotonation/protonation at C3 by two Glu residues. Although the electron density is poor at the 4-, 5-, and 6-positions of the substrates, substrate-enzyme interactions can be deduced from the significant electron density at O6. The O6 possibly interacts with Cys66 via hydrogen bonding, whereas O4 and O5 in d-tagatose and O4 in d-fructose do not undergo hydrogen bonding to the enzyme and are in a hydrophobic environment created by Phe7, Trp15, Trp113, and Phe248. Due to the lack of specific interactions between the enzyme and its substrates at the 4- and 5-positions, P. cichoriid-TE loosely recognizes substrates in this region, allowing it to efficiently catalyze the epimerization of d-tagatose and d-fructose (C4 epimer of d-tagatose) as well. Furthermore, a C3-O3 proton-exchange mechanism for P. cichoriid-TE is suggested by X-ray structural analysis, providing a clear explanation for the regulation of the ionization state of Glu152 and Glu246.     

Crystal structure of Escherichia coli L-arabinose isomerase (ECAI), the putative target of biological tagatose production

Escherichia coli L-arabinose isomerase (ECAI; EC 5.3.1.4) catalyzes the isomerization of L-arabinose to L-ribulose in vivo. This enzyme is also of commercial interest as it catalyzes the conversion of D-galactose to D-tagatose in vitro. The crystal structure of ECAI was solved and refined at 2.6 A resolution. The subunit structure of ECAI is organised into three domains: an N-terminal, a central and a C-terminal domain. It forms a crystallographic trimeric architecture in the asymmetric unit. Packing within the crystal suggests the idea that ECAI can form a hexameric assembly. Previous electron microscopic and biochemical studies supports that ECAI is hexameric in solution. A comparison with other known structures reveals that ECAI adopts a protein fold most similar to E. coli fucose isomerase (ECFI) despite very low sequence identity 9.7%. The structural similarity between ECAI and ECFI with regard to number of domains, overall fold, biological assembly, and active site architecture strongly suggests that the enzymes have functional similarities. Further, the crystal structure of ECAI forms a basis for identifying molecular determinants responsible for isomerization of arabinose to ribulose in vivo and galactose to tagatose in vitro.     

The central cavity from the (alpha/alpha)6 barrel structure of Anabaena sp. CH1 N-acetyl-D-glucosamine 2-epimerase contains two key histidine residues for reversible conversion

N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) catalyzes the reversible epimerization between N-acetyl-D-glucosamine (GlcNAc) and N-acetyl-D-mannosamine (ManNAc). We report here the 2.0 A resolution crystal structure of the GlcNAc 2-epimerase from Anabaena sp. CH1. The structure demonstrates an (alpha/alpha)(6) barrel fold, which shows structural homology with porcine GlcNAc 2-epimerase as well as a number of glycoside hydrolase enzymes and other sugar-metabolizing enzymes. One side of the barrel structure consists of short loops involved in dimer interactions. The other side of the barrel structure is comprised of long loops containing six short beta-sheets, which enclose a putative central active-site pocket. Site-directed mutagenesis of conserved residues near the N-terminal region of the inner alpha helices shows that R57, H239, E308, and H372 are strictly required for activity. E242 and R375 are also essential in catalysis. Based on the structure and kinetic analysis, H239 and H372 may serve as the key active site acid/base catalysts. These results suggest that the (alpha/alpha)(6) barrel represents a steady fold for presenting active-site residues in a cleft at the N-terminal ends of the inner alpha helices, thus forming a fine-tuned catalytic site in GlcNAc 2-epimerase.     

Crystal structure of the RRM domain of poly(A)-specific ribonuclease reveals a novel m(7)G-cap-binding mode

Poly(A)-specific ribonuclease (PARN) is a processive 3′-exoribonuclease involved in the decay of eukaryotic mRNAs. Interestingly, PARN interacts not only with the 3′ end of the mRNA but also with its 5′ end as PARN contains an RRM domain that specifically binds both the poly(A) tail and the 7-methylguanosine (m⁷G) cap. The interaction of PARN with the 5′ cap of mRNAs stimulates the deadenylation activity and enhances the processivity of this reaction. We have determined the crystal structure of the PARN-RRM domain with a bound m⁷G triphosphate nucleotide, revealing a novel binding mode for the m⁷G cap. The structure of the m⁷G binding pocket is located outside of the canonical RNA-binding surface of the RRM domain and differs significantly from that of other m⁷G-cap-binding proteins. The crystal structure also shows a remarkable conformational flexibility of the RRM domain, leading to a perfect exchange of two α-helices with an adjacent protein molecule in the crystal lattice.     

The first structure of a glycoside hydrolase family 61 member, Cel61B from Hypocrea jecorina, at 1.6 A resolution

The glycoside hydrolase (GH) family 61 is a long-recognized, but still recondite, class of proteins, with little known about the activity, mechanism or function of its more than 70 members. The best-studied GH family 61 member, Cel61A of the filamentous fungus Hypocrea jecorina, is known to be an endoglucanase, but it is not clear if this represents the main activity or function of this family in vivo. We present here the first structure for this family, that of Cel61B from H. jecorina. The best-quality crystals were formed in the presence of nickel, and the crystal structure was solved to 1.6 Å resolution using a single-wavelength anomalous dispersion method with nickel as the source of anomalous scatter. Cel61B lacks a carbohydrate-binding module and is a single-domain protein that folds into a twisted β-sandwich. A structure-aided sequence alignment of all GH family 61 proteins identified a highly conserved group of residues on the surface of Cel61B. Within this patch of mostly polar amino acids was a site occupied by the intramolecular nickel hexacoordinately bound in the solved structure. In the Cel61B structure, there is no easily identifiable carbohydrate-binding cleft or pocket or catalytic center of the types normally seen in GHs. A structural comparison search showed that the known structure most similar to Cel61B is that of CBP21 from the Gram-negative soil bacterium Serratia marcescens, a member of the carbohydrate-binding module family 33 proteins. A polar surface patch highly conserved in that structural family has been identified in CBP21 and shown to be involved in chitin binding and in the protein's enhancement of chitinase activities. The analysis of the Cel61B structure is discussed in light of our continuing research to better understand the activities and function of GH family 61.     

Structure and action of the N-oxygenase AurF from Streptomyces thioluteus

Nitro groups are found in a number of bioactive compounds. Most of them arise by a stepwise mono-oxygenation of amino groups. One of the involved enzymes is AurF participating in the biosynthesis of aureothin. Its structure was established at 2.1 A resolution showing a homodimer with a binuclear manganese cluster. The enzyme preparation, which yielded the analyzed crystals, showed activity using in vitro and in vivo assays. Chain fold and cluster are homologous with ribonucleotide reductase subunit R2 and related enzymes. The two manganese ions and an iron content of about 15% were established by anomalous X-ray diffraction. A comparison of the cluster with more common di-iron clusters suggested an additional histidine in the coordination sphere to cause the preference for manganese over iron. There is no oxo-bridge. The substrate p-amino-benzoate was modeled into the active center. The model is supported by mutant activity measurements. It shows the geometry of the reaction and explains the established substrate spectrum.     

Changing the substrate specificity of a chitooligosaccharide oxidase from Fusarium graminearum by model-inspired site-directed mutagenesis

Chitooligosaccharide oxidase (ChitO) catalyzes the oxidation of C1 hydroxyl moieties on chitooligosaccharides and in this way displays a different substrate preference as compared to other known oligosaccharide oxidases. ChitO was identified in the genome of Fusarium graminearum and a structural model revealed that one active site residue (Q268) was likely to be involved in the recognition of the N-acetyl moiety on the chitooligosaccharide substrates. The substrate specificity of wild type ChitO and the Q268R mutant were examined and confirmed that Q268 is indeed involved in N-acetyl recognition.