A versatile cis-prenyltransferase from Methanosarcina mazei catalyzes both C- and O-prenylations.

Polyprenyl groups, products of isoprenoid metabolism, are utilized in peptidoglycan biosynthesis, protein N-glycosylation, and other processes. These groups are formed by cis-prenyltransferases, which use allylic prenyl pyrophosphates as prenyl-donors to catalyze the C-prenylation of the general acceptor substrate, isopentenyl pyrophosphate. Repetition of this reaction forms (Z,E-mixed)-polyprenyl pyrophosphates, which are converted later into glycosyl carrier lipids, such as undecaprenyl phosphate and dolichyl phosphate. MM_0014 from the methanogenic archaeon Methanosarcina mazei is known as a versatile cis-prenyltransferase that accepts both isopentenyl pyrophosphate and dimethylallyl pyrophosphate as acceptor substrates. To learn more about this enzyme's catalytic activity, we determined the X-ray crystal structures of MM_0014 in the presence or absence of these substrates. Surprisingly, one structure revealed a complex with O-prenylglycerol, suggesting that the enzyme catalyzed the prenylation of glycerol contained in the crystallization buffer. Further analyses confirmed that the enzyme could catalyze the O-prenylation of small alcohols, such as 2-propanol, expanding our understanding of the catalytic ability of cis-prenyltransferases.

A Single Amino Acid Mutation Converts (R)-5-Diphosphomevalonate Decarboxylase into a Kinase.

The biosynthesis of isopentenyl diphosphate, a fundamental precursor for isoprenoids, via the mevalonate pathway is completed by diphosphomevalonate decarboxylase. This enzyme catalyzes the formation of isopentenyl diphosphate through the ATP-dependent phosphorylation of the 3-hydroxyl group of (R)-5-diphosphomevalonate followed by decarboxylation coupled with the elimination of the 3-phosphate group. In this reaction, a conserved aspartate residue has been proposed to be involved in the phosphorylation step as the general base catalyst that abstracts a proton from the 3-hydroxyl group. In this study, the catalytic mechanism of this rare type of decarboxylase is re-investigated by structural and mutagenic studies on the enzyme from a thermoacidophilic archaeon Sulfolobus solfataricus The crystal structures of the archaeal enzyme in complex with (R)-5-diphosphomevalonate and adenosine 5'-O-(3-thio)triphosphate or with (R)-5-diphosphomevalonate and ADP are newly solved, and theoretical analysis based on the structure suggests the inability of proton abstraction by the conserved aspartate residue, Asp-281. Site-directed mutagenesis on Asp-281 creates mutants that only show diphosphomevalonate 3-kinase activity, demonstrating that the residue is required in the process of phosphate elimination/decarboxylation, rather than in the preceding phosphorylation step. These results enable discussion of the catalytic roles of the aspartate residue and provide clear proof of the involvement of a long predicted intermediate, (R)-3-phospho-5-diphosphomevalonate, in the reaction of the enzyme.

Mannose-recognition mutant of the galactose/N-acetylgalactosamine-specific C-type lectin CEL-I engineered by site-directed mutagenesis.

BACKGROUND: CEL-I is a galactose/N-acetylgalactosamine-specific C-type lectin isolated from the sea cucumber Cucumaria echinata. Its carbohydrate-binding site contains a QPD (Gln-Pro-Asp) motif, which is generally recognized as the galactose specificity-determining motif in the C-type lectins. In our previous study, replacement of the QPD motif by an EPN (Glu-Pro-Asn) motif led to a weak binding affinity for mannose. Therefore, we examined the effects of an additional mutation in the carbohydrate-binding site on the specificity of the lectin. METHODS: Trp105 of EPN-CEL-I was replaced by a histidine residue using site-directed mutagenesis, and the binding affinity of the resulting mutant, EPNH-CEL-I, was examined by sugar-polyamidoamine dendrimer assay, isothermal titration calorimetry, and glycoconjugate microarray analysis. Tertiary structure of the EPNH-CEL-I/mannose complex was determined by X-ray crystallographic analysis. RESULTS: Sugar-polyamidoamine dendrimer assay and glycoconjugate microarray analysis revealed a drastic change in the specificity of EPNH-CEL-I from galactose/N-acetylgalactosamine to mannose. The association constant of EPNH-CEL-I for mannose was determined to be 3.17×10(3) M(-1) at 25°C. Mannose specificity of EPNH-CEL-I was achieved by stabilization of the binding of mannose in a correct orientation, in which the EPN motif can form proper hydrogen bonds with 3- and 4-hydroxy groups of the bound mannose. CONCLUSIONS: Specificity of CEL-I can be engineered by mutating a limited number of amino acid residues in addition to the QPD/EPN motifs. GENERAL SIGNIFICANCE: Versatility of the C-type carbohydrate-recognition domain structure in the recognition of various carbohydrate chains could become a promising platform to develop novel molecular recognition proteins.

Effects of amino acid mutations in the pore-forming domain of the hemolytic lectin CEL-III.

The hemolytic lectin CEL-III forms transmembrane pores in the membranes of target cells. A study on the effect of site-directed mutation at Lys405 in domain 3 of CEL-III indicated that replacements of this residue by relatively smaller residues lead to a marked increase in hemolytic activity, suggesting that moderately destabilizing domain 3 facilitates formation of transmembrane pores through conformational changes.

In Vivo Formation of the Protein Disulfide Bond That Enhances the Thermostability of Diphosphomevalonate Decarboxylase, an Intracellular Enzyme from the Hyperthermophilic Archaeon Sulfolobus solfataricus.

UNLABELLED: In the present study, the crystal structure of recombinant diphosphomevalonate decarboxylase from the hyperthermophilic archaeon Sulfolobus solfataricus was solved as the first example of an archaeal and thermophile-derived diphosphomevalonate decarboxylase. The enzyme forms a homodimer, as expected for most eukaryotic and bacterial orthologs. Interestingly, the subunits of the homodimer are connected via an intersubunit disulfide bond, which presumably formed during the purification process of the recombinant enzyme expressed in Escherichia coli. When mutagenesis replaced the disulfide-forming cysteine residue with serine, however, the thermostability of the enzyme was significantly lowered. In the presence of ß-mercaptoethanol at a concentration where the disulfide bond was completely reduced, the wild-type enzyme was less stable to heat. Moreover, Western blot analysis combined with nonreducing SDS-PAGE of the whole cells of S. solfataricus proved that the disulfide bond was predominantly formed in the cells. These results suggest that the disulfide bond is required for the cytosolic enzyme to acquire further thermostability and to exert activity at the growth temperature of S. solfataricus. IMPORTANCE: This study is the first report to describe the crystal structures of archaeal diphosphomevalonate decarboxylase, an enzyme involved in the classical mevalonate pathway. A stability-conferring intersubunit disulfide bond is a remarkable feature that is not found in eukaryotic and bacterial orthologs. The evidence that the disulfide bond also is formed in S. solfataricus cells suggests its physiological importance.

Hemolytic lectin CEL-III heptamerizes via a large structural transition from α-helices to a ß-barrel during the transmembrane pore formation process.

CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-binding domains (domains 1 and 2) and one oligomerization domain (domain 3). After binding to the cell surface carbohydrate chains through domains 1 and 2, domain 3 self-associates to form transmembrane pores, leading to cell lysis or death, which resembles other pore-forming toxins of diverse organisms. To elucidate the pore formation mechanism of CEL-III, the crystal structure of the CEL-III oligomer was determined. The CEL-III oligomer has a heptameric structure with a long ß-barrel as a transmembrane pore. This ß-barrel is composed of 14 ß-strands resulting from a large structural transition of α-helices accommodated in the interface between domains 1 and 2 and domain 3 in the monomeric structure, suggesting that the dissociation of these α-helices triggered their structural transition into a ß-barrel. After heptamerization, domains 1 and 2 form a flat ring, in which all carbohydrate-binding sites remain bound to cell surface carbohydrate chains, stabilizing the transmembrane ß-barrel in a position perpendicular to the plane of the lipid bilayer.

Structural insights into the low pH adaptation of a unique carboxylesterase from Ferroplasma: altering the pH optima of two carboxylesterases.

To investigate the mechanism for low pH adaptation by a carboxylesterase, structural and biochemical analyses of EstFa_R (a recombinant, slightly acidophilic carboxylesterase from Ferroplasma acidiphilum) and SshEstI (an alkaliphilic carboxylesterase from Sulfolobus shibatae DSM5389) were performed. Although a previous proteomics study by another group showed that the enzyme purified from F. acidiphilum contained an iron atom, EstFa_R did not bind to iron as analyzed by inductively coupled plasma MS and isothermal titration calorimetry. The crystal structures of EstFa_R and SshEstI were determined at 1.6- and 1.5-Å resolutions, respectively. EstFa_R had a catalytic triad with an extended hydrogen bond network that was not observed in SshEstI. Quadruple mutants of both proteins were created to remove or introduce the extended hydrogen bond network. The mutation on EstFa_R enhanced its catalytic efficiency and gave it an alkaline pH optimum, whereas the mutation on SshEstI resulted in opposite effects (i.e. a decrease in the catalytic efficiency and a downward shift in the optimum pH). Our experimental results suggest that the low pH optimum of EstFa_R activity was a result of the unique extended hydrogen bond network in the catalytic triad and the highly negatively charged surface around the active site. The change in the pH optimum of EstFa_R happened simultaneously with a change in the catalytic efficiency, suggesting that the local flexibility of the active site in EstFa_R could be modified by quadruple mutation. These observations may provide a novel strategy to elucidate the low pH adaptation of serine hydrolases.

Identification of the amino acid residues involved in the hemolytic activity of the Cucumaria echinata lectin CEL-III.

BACKGROUND: CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata that shows Ca(2+)-dependent Gal/GalNAc-binding specificity. This lectin is composed of two carbohydrate-recognition domains (domains 1 and 2) and an oligomerization domain (domain 3) that facilitates CEL-III assembly in the target cell membrane to form ion-permeable pores. METHODS: Several amino acid residues in domain 3 were replaced by alanine, and hemolytic activity of the mutants was examined. RESULTS: K344A, K351A, K405A, K420A and K425A showed marked increases in activity. In particular, K405A had activity that was 360-fold higher than the wild-type recombinant CEL-III and 3.6-fold higher than the native protein purified from sea cucumber. Since these residues appear to play roles in the stabilization of domain 3 through ionic and hydrogen bonding interactions with other residues, the mutations of these residues presumably lead to destabilization of domain 3, which consequently induces the oligomerization of the protein through association of domain 3 in the membrane. In contrast, K338A, R378A and R408A mutants exhibited a marked decrease in hemolytic activity. Since these residues are located on the surface of domain 3 without significant interactions with other residue, they may be involved in the interaction with components of the target cell membrane. CONCLUSIONS: Several amino acid residues, especially basic residues, are found to be involved in the hemolytic activity as well as the oligomerization ability of CEL-III. GENERAL SIGNIFICANCE: The results provide important clues to the membrane pore-forming mechanism of CEL-III, which is also related to that of bacterial pore-forming toxins.

Manno-oligosaccharide-binding ability of mouse RegIV/GST-fusion protein evaluated by complex formation with the carbohydrate-containing polyamidoamine dendrimer.

The carbohydrate-binding properties of the C-type lectin-like mouse RegIV and glutathione S-transferase-fusion protein (GST-mRegIV) were examined using carbohydrate-containing polyamidoamine dendrimers (PD). GST-mRegIV showed affinity for mannan- and manno-oligosaccharide containing PD. Binding was inhibited by manno-oligosaccharides but not by mannose or other tested carbohydrates, suggesting that the binding site may have an extended structure in contrast with typical C-type lectins.

Covalent modification of reduced flavin mononucleotide in type-2 isopentenyl diphosphate isomerase by active-site-directed inhibitors.

Evidence for an unusual catalysis of protonation/deprotonation by a reduced flavin mononucleotide cofactor is presented for type-2 isopentenyl diphosphate isomerase (IDI-2), which catalyzes isomerization of the two fundamental building blocks of isoprenoid biosynthesis, isopentenyl diphosphate and dimethylallyl diphosphate. The covalent adducts formed between irreversible mechanism-based inhibitors, 3-methylene-4-penten-1-yl diphosphate or 3-oxiranyl-3-buten-1-yl diphosphate, and the flavin cofactor were investigated by X-ray crystallography and UV-visible spectroscopy. Both the crystal structures of IDI-2 binding the flavin-inhibitor adduct and the UV-visible spectra of the adducts indicate that the covalent bond is formed at C4a of flavin rather than at N5, which had been proposed previously. In addition, the high-resolution crystal structures of IDI-2-substrate complexes and the kinetic studies of new mutants confirmed that only the flavin cofactor can catalyze protonation of the substrates and suggest that N5 of flavin is most likely to be involved in proton transfer. These data provide support for a mechanism where the reduced flavin cofactor acts as a general acid/base catalyst and helps stabilize the carbocationic intermediate formed by protonation.

Carbohydrate-binding ability of a recombinant protein containing the DM9 motif from Drosophila melanogaster.

Proteins containing DM9 motifs, which were originally identified in the Drosophila melanogaster genome, are widely distributed in various organisms and are assumed to be involved in their innate immune response. In this study, we produced a recombinant protein of CG13321 (rCG13321) from D. melanogaster, which consists of four DM9 motifs, in Escherichia coli cells. In affinity chromatography using a mannose-immobilized column, rCG13321 exhibited mannose-binding ability and was separated into high-affinity and low-affinity fractions, named HA and LA, respectively, based on its binding ability to the column. In addition to having a higher affinity for the column, HA exhibited self-oligomerization ability, suggesting slight differences in tertiary structure. Both LA and HA showed hemagglutinating activity and were able to agglutinate an oligomannose-containing dendrimer, indicating that they have multiple carbohydrate-binding sites. Glycan array analysis suggested that rCG13321 primarily recognizes d-mannose and d-rhamnose through hydrogen bonding with the 2-, 3- and 4-hydroxy groups. Isothermal titration calorimetry demonstrated that rCG13321 has a comparable affinity to typical lectins. These findings suggest that CG13321 functions as a carbohydrate-binding protein or lectin that recognizes mannose and related carbohydrate-containing molecules on the surface of foreign organisms as a pattern recognition molecule.

Lowering pH optimum of activity of SshEstI, a slightly alkaliphilic archaeal esterase of the hormone-sensitive lipase family.

SshEstI, a carboxylesterase from the thermoacidophilic archaeon Saccharolobus shibatae, is a member of the hormone-sensitive lipase family that displays slightly alkaliphilic activity with an optimum activity at pH 8.0. In this study, three distinct strategies were explored to confer acidophilic properties to SshEstI. The first strategy involved engineering the oxyanion hole by replacing Gly81 with serine or aspartic acid. The G81S mutant showed optimum activity at pH 7.0, whereas the aspartic acid mutant (G81D) rendered the enzyme slightly acidophilic with optimum activity observed at pH 6.0; however, kcat and kcat/Km values were reduced by these substitutions. The second strategy involved examining the effects of surfactant additives on the pH-activity profiles of SshEstI. The results showed that cetyltrimethylammonium bromide (CTAB) enhanced wild-type enzyme (WT) activity at acidic pH values. In the presence of 0.1 mM CTAB, G81S and G81D were acidophilic enzymes with optimum activity at pH 6.0 and 4.0, respectively, although their enzyme activities were low. The third strategy involved engineering the active site to resemble that of kumamolisin-As (kuma-As), an acidophilic peptidase of the sedolisin family. The catalytic triad of kuma-As was exchanged into SshEstI using site-directed mutagenesis. X-ray crystallographic analysis of the mutants (H274D and H274E) revealed that the potential hydrogen donor-acceptor distances around the active site of WT were fully maintained in these mutants. However, these mutants were inactive at pH 4-8.

Crystallization and preliminary crystallographic study of oligomers of the haemolytic lectin CEL-III from the sea cucumber Cucumaria echinata.

CEL-III is a Ca(2+)-dependent haemolytic lectin isolated from the marine invertebrate Cucumaria echinata. This lectin binds to Gal/GalNAc-containing carbohydrate chains on the cell surface and, after conformational changes, oligomerizes to form ion-permeable pores in cell membranes. CEL-III also forms soluble oligomers similar to those formed in cell membranes upon binding of specific carbohydrates in high-pH and high-salt solutions. These soluble and membrane CEL-III oligomers were crystallized and X-ray diffraction data were collected. Crystals of soluble oligomers and membrane oligomers diffracted X-rays to 3.3 and 4.2 Å resolution, respectively, using synchrotron radiation and the former was found to belong to space group C2. Self-rotation functional analysis of the soluble oligomer crystal suggested that it might be composed of heptameric CEL-III.

Effects of detergents on the oligomeric structures of hemolytic lectin CEL-III as determined by small-angle X-ray scattering.

Hemolytic lectin CEL-III isolated from the sea cucumber Cucumaria echinata forms transmembrane pores by self-oligomerization in target cell membranes. It also formed soluble oligomers in aqueous solution upon binding with specific carbohydrates under conditions of high pH and a high salt concentration. The size of the soluble CEL-III oligomers decreased when treated with detergents such as Triton X-100 and SDS. Small-angle X-ray scattering measurements suggested that the dissociated unit of the oligomer was a tightly associated CEL-III heptamer. Without detergents in solution, these heptamers further assembled into larger 21mer oligomers, comprising three heptamers held together by relatively weak hydrophobic interactions.

Functional Diversity of Novel Lectins with Unique Structural Features in Marine Animals.

Due to their remarkable structural diversity, glycans play important roles as recognition molecules on cell surfaces of living organisms. Carbohydrates exist in numerous isomeric forms and can adopt diverse structures through various branching patterns. Despite their relatively small molecular weights, they exhibit extensive structural diversity. On the other hand, lectins, also known as carbohydrate-binding proteins, not only recognize and bind to the diverse structures of glycans but also induce various biological reactions based on structural differences. Initially discovered as hemagglutinins in plant seeds, lectins have been found to play significant roles in cell recognition processes in higher vertebrates. However, our understanding of lectins in marine animals, particularly marine invertebrates, remains limited. Recent studies have revealed that marine animals possess novel lectins with unique structures and glycan recognition mechanisms not observed in known lectins. Of particular interest is their role as pattern recognition molecules in the innate immune system, where they recognize the glycan structures of pathogens. Furthermore, lectins serve as toxins for self-defense against foreign enemies. Recent discoveries have identified various pore-forming proteins containing lectin domains in fish venoms and skins. These proteins utilize lectin domains to bind target cells, triggering oligomerization and pore formation in the cell membrane. These findings have spurred research into the new functions of lectins and lectin domains. In this review, we present recent findings on the diverse structures and functions of lectins in marine animals.

Mannose oligosaccharide recognition of CGL1, a mannose-specific lectin containing DM9 motifs from Crassostrea gigas, revealed by X-ray crystallographic analysis.

CGL1 is a mannose-specific lectin isolated from the Pacific oyster Crassostrea gigas, and it belongs to the DM9 domain protein family. Each subunit of the CGL1 dimer consists of a tandem repeat of DM9 motifs, which were originally found in the Drosophila melanogaster genome. The CGL1 protomer contains two carbohydrate-binding sites: a high-affinity site A and a low-affinity site B. An assay using dendrimers containing oligomannose from yeast (Saccharomyces cerevisiae) revealed that CGL1 exhibited significantly higher affinity for mannotetraose (Man4) compared to mannobiose (Man2) and mannotriose (Man3). To investigate its oligomannose-recognition mechanism, X-ray crystallographic analyses of CGL1/oligomannose complexes were performed. In the CGL1/Man2 and CGL1/Man3 complexes, Manα1-2Man and Manα1-2Manα1-2Man, respectively, were primarily bound to site A, interacting with the non-reducing mannose residue. On the other hand, in the CGL1/Man4 crystal, Man4 (Manα1-2Manα1-2Manα1-6Man) was bound at both site A and site B at the non-reducing and reducing ends, thus linking adjacent CGL1 molecules with crystallographic symmetry. These findings suggest that CGL1 can recognize both the non-reducing and reducing mannose residues of mannose oligosaccharides at its two distinct carbohydrate-binding sites. This enables efficient complex formation, making CGL1 a pattern-recognition molecule capable of recognizing diverse structures of mannose-containing carbohydrate chains.

Substrate-induced change in the quaternary structure of type 2 isopentenyl diphosphate isomerase from Sulfolobus shibatae.

Type 2 isopentenyl diphosphate isomerase catalyzes the interconversion between two active units for isoprenoid biosynthesis, i.e., isopentenyl diphosphate and dimethylallyl diphosphate, in almost all archaea and in some bacteria, including human pathogens. The enzyme is a good target for discovery of antibiotics because it is essential for the organisms that use only the mevalonate pathway to produce the active isoprene units and because humans possess a nonhomologous isozyme, type 1 isopentenyl diphosphate isomerase. However, type 2 enzymes were reportedly inhibited by mechanism-based drugs for the type 1 enzyme due to their surprisingly similar reaction mechanisms. Thus, a different approach is now required to develop new inhibitors specific to the type 2 enzyme. X-ray crystallography and gel filtration chromatography revealed that the enzyme from a thermoacidophilic archaeon, Sulfolobus shibatae, is in the octameric state at a high concentration. Interestingly, a part of the regions that are involved in the substrate binding in the previously reported tetrameric structures is integral to the formation of the tetramer-tetramer interface in the substrate-free octameric structure. Site-directed mutagenesis at such regions resulted in stabilization of the tetramer. Small-angle X-ray scattering, tryptophan fluorescence, and dynamic light scattering analyses showed that substrate binding causes the dissociation of an octamer into tetramers. This property, i.e., incompatibility between octamer formation and substrate binding, might provide clues to develop new specific inhibitors of the archaeal enzyme.

Galactose recognition by a tetrameric C-type lectin, CEL-IV, containing the EPN carbohydrate recognition motif.

CEL-IV is a C-type lectin isolated from a sea cucumber, Cucumaria echinata. This lectin is composed of four identical C-type carbohydrate-recognition domains (CRDs). X-ray crystallographic analysis of CEL-IV revealed that its tetrameric structure was stabilized by multiple interchain disulfide bonds among the subunits. Although CEL-IV has the EPN motif in its carbohydrate-binding sites, which is known to be characteristic of mannose binding C-type CRDs, it showed preferential binding of galactose and N-acetylgalactosamine. Structural analyses of CEL-IV-melibiose and CEL-IV-raffinose complexes revealed that their galactose residues were recognized in an inverted orientation compared with mannose binding C-type CRDs containing the EPN motif, by the aid of a stacking interaction with the side chain of Trp-79. Changes in the environment of Trp-79 induced by binding to galactose were detected by changes in the intrinsic fluorescence and UV absorption spectra of WT CEL-IV and its site-directed mutants. The binding specificity of CEL-IV toward complex oligosaccharides was analyzed by frontal affinity chromatography using various pyridylamino sugars, and the results indicate preferential binding to oligosaccharides containing Galß1-3/4(Fucα1-3/4)GlcNAc structures. These findings suggest that the specificity for oligosaccharides may be largely affected by interactions with amino acid residues in the binding site other than those determining the monosaccharide specificity.

An assay for carbohydrate-binding activity of lectins using polyamidoamine dendrimer conjugated with carbohydrates.

The carbohydrate-binding activity of lectins was examined using polyamidoamine dendrimer conjugated with carbohydrates (sugar-PD). When a C-type lectin, CEL-IV, was mixed with melibiose-PD, large complexes with a diameter of about 1 µm were formed. Changes in the amount of CEL-IV/melibiose-PD complex as an indication of lectin activity were measured sensitively by Rayleigh scattering. The carbohydrate specificity of the lectin was determined on the basis of inhibition of complex-formation by individual carbohydrates. It is suggested that various lectins can also be measured using sugar-PDs to which different carbohydrates are attached.

Biological and immunological characteristics of hepatitis E virus-like particles based on the crystal structure.

Hepatitis E virus (HEV) is a causative agent of acute hepatitis. The crystal structure of HEV-like particles (HEV-LP) consisting of capsid protein was determined at 3.5-A resolution. The capsid protein exhibited a quite different folding at the protruding and middle domains from the members of the families of Caliciviridae and Tombusviridae, while the shell domain shared the common folding. Tyr-288 at the 5-fold axis plays key roles in the assembly of HEV-LP, and aromatic amino acid residues are well conserved among the structurally related viruses. Mutational analyses indicated that the protruding domain is involved in the binding to the cells susceptive to HEV infection and has some neutralization epitopes. These structural and biological findings are important for understanding the molecular mechanisms of assembly and entry of HEV and also provide clues in the development of preventive and prophylactic measures for hepatitis E.