The three domains of a bacterial sialidase: a beta-propeller, an immunoglobulin module and a galactose-binding jelly-roll.

BACKGROUND: Sialidases, or neuraminidases, have been implicated in the pathogenesis of many diseases, but are also produced by many non-pathogenic bacteria. Bacterial sialidases are very variable in size, often possessing domains in addition to the catalytic domain. The sialidase from the non-pathogenic soil bacterium Micromonospora viridifaciens is secreted in two forms with molecular weights of 41 kDa or 68 kDa, depending on the nature of the carbohydrate used to induce expression. RESULTS: We report here the X-ray crystal structures of the 41 kDa and 68 kDa forms of the sialidase from M. viridifaciens at 1.8 A and 2.5 A resolution respectively. In addition, we report a complex of the 41 kDa form with an inhibitor at 2.0 A resolution, and a complex of the 68 kDa form with galactose at 2.5 A. The 41 kDa form shows the canonical sialidase beta-propeller fold. The 68 kDa form possesses two additional domains, one with an immunoglobulin-like fold that serves as a linker to the second, which is homologous to the galactose-binding domain of a fungal galactose oxidase. CONCLUSIONS: The presence of the additional carbohydrate-binding domain in the 68 kDa form of the bacterial sialidase reported here is a further example of a combination of carbohydrate binding and cleaving domains which we observed in the sialidase from Vibrio cholerae. This dual function may be common, but only to other bacterial and parasitic sialidases, but also to other secreted glycosidases involved in pathogenesis. The bacterium may have acquired both the immunoglobulin module and the galactose-binding module from eukaryotes, as the enzyme shows a remarkable similarity to a fungal galactose oxidase which possesses similar domains performing different functions and assembled in a different order.

Crystal structure of Vibrio cholerae neuraminidase reveals dual lectin-like domains in addition to the catalytic domain.

BACKGROUND: Vibrio cholerae neuraminidase is part of a mucinase complex which may function in pathogenesis by degrading the mucin layer of the gastrointestinal tract. The neuraminidase, which has been the target of extensive inhibitor studies, plays a subtle role in the pathology of the bacterium, by processing higher order gangliosides to GM1, the receptor for cholera toxin. RESULTS: We report here the X-ray crystal structure of V. cholerae neuraminidase at 2.3 A resolution. The 83 kDa enzyme folds into three distinct domains. The central catalytic domain has the canonical neuraminidase beta-propeller fold, and is flanked by two domains which possess identical legume lectin-like topologies but without the usual metal-binding loops. The active site has many features in common with other viral and bacterial neuraminidases but, uniquely, has an essential Ca2+ ion which plays a crucial structural role. CONCLUSIONS: The environment of the small intestine requires V. cholerae to secrete several adhesins, and it is known that its neuraminidase can bind to cell surfaces, and remain active. The unexpected lectin-like domains possibly mediate this attachment. These bacterial lectin folds represent additional members of a growing lectin superfamily.

The crystal structure of glucose dehydrogenase from Thermoplasma acidophilum.

BACKGROUND: The archaea are a group of organisms distinct from bacteria and eukaryotes. Structures of proteins from archaea are of interest because they function in extreme environments and because structural studies may reveal evolutionary relationships between proteins. The enzyme glucose dehydrogenase from the thermophilic archaeon Thermoplasma acidophilum is of additional interest because it is involved in an unusual pathway of sugar metabolism. RESULTS: We have determined the crystal structure of this glucose dehydrogenase to 2.9 A resolution. The monomer comprises a central nucleotide-binding domain, common to other nucleotide-binding dehydrogenases, flanked by the catalytic domain. Unexpectedly, we observed significant structural homology between the catalytic domain of horse liver alcohol dehydrogenase and T. acidophilum glucose dehydrogenase. CONCLUSIONS: The structural homology between glucose dehydrogenase and alcohol dehydrogenase suggests an evolutionary relationship between these enzymes. The quaternary structure of glucose dehydrogenase may provide a model for other tetrameric alcohol/polyol dehydrogenases. The predicted mode of nucleotide binding provides a plausible explanation for the observed dual-cofactor specificity, the molecular basis of which can be tested by site-directed mutagenesis.

The structures of Salmonella typhimurium LT2 neuraminidase and its complexes with three inhibitors at high resolution.

The structure of Salmonella typhimurium LT2 neuraminidase (STNA) is reported here to a resolution of 1.6 angstroms together with the structures of three complexes of STNA with different inhibitors. The first is 2-deoxy-2,3-dehydro-N-acetyl-neuraminic acid (Neu5Ac2en or DANA), the second and third are phosphonate derivatives of N-acetyl-neuraminic acid (NANA) which have phosphonate groups at the C2 position equatorial (ePANA) and axial (aPANA) to the plane of the sugar ring. The complex structures are at resolutions of 1.6 angstroms, 1.6 angstroms and 1.9 angstroms, respectively. These analyses show the STNA active site to be topologically inflexible and the interactions to be dominated by the arginine triad, with the pyranose rings of the inhibitors undergoing distortion to occupy the space available. Solvent structure differs only around the third phosphonate oxygen, which attracts a potassium ion. The STNA structure is topologically identical to the previously reported influenza virus neuraminidase structures, although very different in detail; the root-mean-square (r.m.s) deviation for 210 C alpha positions considered equivalent is 2.28 angstroms (out of a total of 390 residues in influenza and 381 in STNA). The active site residues are more highly conserved, in that both the viral and bacterial structures contain an arginine triad, a hydrophobic pocket, a tyrosine and glutamic acid residue at the base of the site and a potential proton-donating aspartic acid. However, differences in binding to O4 and to the glycerol side-chain may reflect the different kinetics employed by the two enzymes.

The predicted structure of photopexin from Photorhabdus shows the first haemopexin-like motif in prokaryotes.

The insect pathogenic bacterium Photorhabdus luminescens secretes several insecticidal high molecular mass 'toxin complexes'. Analysis of the putative pathogenicity island surrounding the toxin complex a (tca) locus revealed two open reading frames (ORFs) of unknown function. The predicted protein sequences of these ORFs show a repeated motif similar to those found in the vertebrate haem scavenging molecule haemopexin, limunectin (a phosphocholine binding protein from Limulus) and the C-terminal domains of matrix metalloproteinases (MMPs) (where they are thought to be important for cell attachment and adhesion). We have therefore named the operon photopexin AB and the putative encoded proteins 'photopexins' A and B (PpxA and PpxB). The predicted amino acid sequence of PpxA was modelled onto the crystal structure of a MMP. Our model predicts not only that PpxA and PpxB have beta-propeller domains but also that each haemopexin-like repeat corresponds to one blade of a propeller, suggesting the limunectin structure itself may also contain two or three such haemopexin-like propellers. The overall structure of PpxA has striking similarity to that of haemopexin suggesting that it may be used by the bacterium to scavenge iron containing compounds from insects. The implications for the potential role of Ppx proteins in pathogenicity are discussed. This is the first finding of a haemopexin-like repeat outside plants and animals.

Crystal structure of the multifunctional paramyxovirus hemagglutinin-neuraminidase.

Paramyxoviruses are the main cause of respiratory disease in children. One of two viral surface glycoproteins, the hemagglutinin-neuraminidase (HN), has several functions in addition to being the major surface antigen that induces neutralizing antibodies. Here we present the crystal structures of Newcastle disease virus HN alone and in complex with either an inhibitor or with the beta-anomer of sialic acid. The inhibitor complex reveals a typical neuraminidase active site within a beta-propeller fold. Comparison of the structures of the two complexes reveal differences in the active site, suggesting that the catalytic site is activated by a conformational switch. This site may provide both sialic acid binding and hydrolysis functions since there is no evidence for a second sialic acid binding site in HN. Evidence for a single site with dual functions is examined and supported by mutagenesis studies. The structure provides the basis for the structure-based design of inhibitors for a range of paramyxovirus-induced diseases.

Crystallization of Newcastle disease virus hemagglutinin-neuraminidase glycoprotein.

The hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus was isolated by cleaving HN (cHN) from reconstituted virosome with chymotrypsin. N-terminal sequence analysis of the purified cHN showed that chymotrypsin cleavage had occurred at amino acid 123, freeing the C-terminal 454 amino acids. The purified cHN retained its neuraminidase and receptor binding activities and reacted with specific monoclonal antibodies, showing that the isolated cHN was biologically and antigenically functional. The crystals of the cHN were obtained in acetate buffer (pH 4.6) containing polyethylene glycol 3350 and ammonium sulfate and belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimension of approximately a = 72 A, b = 78 A, and c = 198 A. Crystals of cHN grown in the presence of sialic acid (Neu5Ac) were grown in HEPES buffer (pH 6.2) containing polyethylene glycol 3350 and belong to the hexagonal space groups P6(1) or P6(5) with unit cell dimensions of a = b = 137.5 A and c = 116.6A. The orthorhombic crystals produced in this study diffract X rays to at least 2.0-A resolution, thereby setting the stage for the solution of the three-dimensional structure of the HN glycoprotein of a paramyxovirus.

Crystal structure of a bacterial sialidase (from Salmonella typhimurium LT2) shows the same fold as an influenza virus neuraminidase.

Sialidases (EC 3.2.1.18 or neuraminidases) remove sialic acid from sialoglycoconjugates, are widely distributed in nature, and have been implicated in the pathogenesis of many diseases. The three-dimensional structure of influenza virus sialidase is known, and we now report the three-dimensional structure of a bacterial sialidase, from Salmonella typhimurium LT2, at 2.0-A resolution and the structure of its complex with the inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid at 2.2-A resolution. The viral enzyme is a tetramer; the bacterial enzyme, a monomer. Although the monomers are of similar size (approximately 380 residues), the sequence similarity is low (approximately 15%). The viral enzyme contains at least eight disulfide bridges, conserved in all strains, and binds Ca2+, which enhances activity; the bacterial enzyme contains one disulfide and does not bind Ca2+. Comparison of the two structures shows a remarkable similarity both in the general fold and in the spatial arrangement of the catalytic residues. However, an rms fit of 3.1 A between 264 C alpha atoms of the S. typhimurium enzyme and those from an influenza A virus reflects some major differences in the fold. In common with the viral enzyme, the bacterial enzyme active site consists of an arginine triad, a hydrophobic pocket, and a key tyrosine and glutamic acid, but differences in the interactions with the O4 and glycerol groups of the inhibitor reflect differing kinetics and substrate preferences of the two enzymes. The repeating "Asp-box" motifs observed among the nonviral sialidase sequences occur at topologically equivalent positions on the outside of the structure. Implications of the structure for the catalytic mechanism, evolution, and secretion of the enzyme are discussed.