Crystal structures of 4-alpha-glucanotransferase from Thermococcus litoralis and its complex with an inhibitor

Thermococcus litoralis 4-alpha-glucanotransferase (TLGT) belongs to glucoside hydrolase family 57 and catalyzes the disproportionation of amylose and the formation of large cyclic alpha-1,4-glucan (cycloamylose) from linear amylose. We determined the crystal structure of TLGT with and without an inhibitor, acarbose. TLGT is composed of two domains: an N-terminal domain (domain I), which contains a (beta/alpha)7 barrel fold, and a C-terminal domain (domain II), which has a twisted beta-sandwich fold. In the structure of TLGT complexed with acarbose, the inhibitor was bound at the cleft within domain I, indicating that domain I is a catalytic domain of TLGT. The acarbose-bound structure also clarified that Glu123 and Asp214 were the catalytic nucleophile and acid/base catalyst, respectively, and revealed the residues involved in substrate binding. It seemed that TLGT produces large cyclic glucans by preventing the production of small cyclic glucans by steric hindrance, which is achieved by three lids protruding into the active site cleft, as well as an extended active site cleft. Interestingly, domain I of TLGT shares some structural features with the catalytic domain of Golgi alpha-mannosidase from Drosophila melanogaster, which belongs to glucoside hydrolase family 38. Furthermore, the catalytic residue of the two enzymes is located in the same position. These observations suggest that families 57 and 38 evolved from a common ancestor.     

Osmoregulated periplasmic glucan synthesis is required for Erwinia chrysanthemi pathogenicity

Erwinia chrysanthemi is a phytopathogenic enterobacterium causing soft rot disease in a wide range of plants. Osmoregulated periplasmic glucans (OPGs) are intrinsic components of the gram-negative bacterial envelope. We cloned the opgGH operon of E. chrysanthemi, encoding proteins involved in the glucose backbone synthesis of OPGs, by complementation of the homologous locus mdoGH of Escherichia coli. OpgG and OpgH show a high level of similarity with MdoG and MdoH, respectively, and mutations in the opgG or opgH gene abolish OPG synthesis. The opg mutants exhibit a pleiotropic phenotype, including overproduction of exopolysaccharides, reduced motility, bile salt hypersensitivity, reduced protease, cellulase, and pectate lyase production, and complete loss of virulence. Coinoculation experiments support the conclusion that OPGs present in the periplasmic space of the bacteria are necessary for growth in the plant host.     

Crystal structure of a natural circularly permuted jellyroll protein: 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes

The 1,3-1,4-beta-D-glucanase from Fibrobacter succinogenes (Fsbeta-glucanase) is classified as one of the family 16 glycosyl hydrolases. It hydrolyzes the glycosidic bond in the mixed-linked glucans containing beta-1,3- and beta-1,4-glycosidic linkages. We constructed a truncated form of recombinant Fsbeta-glucanase containing the catalytic domain from amino acid residues 1-258, which exhibited a higher thermal stability and enzymatic activity than the full-length enzyme. The crystal structure of the truncated Fsbeta-glucanase was solved at a resolution of 1.7A by the multiple wavelength anomalous dispersion (MAD) method using the anomalous signals from the seleno-methionine-labeled protein. The overall topology of the truncated Fsbeta-glucanase consists mainly of two eight-stranded anti-parallel beta-sheets arranged in a jellyroll beta-sandwich, similar to the fold of many glycosyl hydrolases and carbohydrate-binding modules. Sequence comparison with other bacterial glucanases showed that Fsbeta-glucanase is the only naturally occurring circularly permuted beta-glucanase with reversed sequences. Structural comparison shows that the engineered circular-permuted Bacillus enzymes are more similar to their parent enzymes with which they share approximately 70% sequence identity, than to the naturally occurring Fsbeta-glucanase of similar topology with 30% identity. This result suggests that protein structure relies more on sequence identity than topology. The high-resolution structure of Fsbeta-glucanase provides a structural rationale for the different activities obtained from a series of mutant glucanases and a basis for the development of engineered enzymes with increased activity and structural stability.     

Identification of mdoD, an mdoG paralog which encodes a twin-arginine-dependent periplasmic protein that controls osmoregulated periplasmic glucan backbone structures

Osmoregulated periplasmic glucans (OPGs) of Escherichia coli are anionic and highly branched oligosaccharides that accumulate in the periplasmic space in response to low osmolarity of the medium. The glucan length, ranging from 5 to 12 glucose residues, is under strict control. Two genes that form an operon, mdoGH, govern glucose backbone synthesis. The new gene mdoD, which appears to be a paralog of mdoG, was characterized in this study. Cassette inactivation of mdoD resulted in production of OPGs with a higher degree of polymerization, indicating that OpgD, the mdoD product (according to the new nomenclature), controls the glucose backbone structures. OpgD secretion depends on the Tat secretory pathway. Orthologs of the mdoG and mdoD genes are found in various proteobacteria. Most of the OpgD orthologs exhibit a Tat-dependent secretion signal, while most of the OpgG orthologs are Sec dependent.     

Structure of the bacteriophage phi KZ lytic transglycosylase gp144

Lytic transglycosylases are enzymes that act on the peptidoglycan of bacterial cell walls. They cleave the glycosidic linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anhydromuramoyl product. The x-ray structure of the lytic transglycosylase gp144 from the Pseudomonas bacteriophage phi KZ has been determined to 2.5-A resolution. This protein is probably employed by the bacteriophage in the late stage of the virus reproduction cycle to destroy the bacterial cell wall to release the phage progeny. phi KZ gp144 is a 260-residue alpha-helical protein composed of a 70-residue N-terminal cell wall-binding domain and a C-terminal catalytic domain. The fold of the N-terminal domain is similar to the peptidoglycan-binding domain from Streptomyces albus G D-Ala-D-Ala carboxypeptidase and to the N-terminal prodomain of human metalloproteinases that act on extracellular matrices. The C-terminal catalytic domain of gp144 has a structural similarity to the catalytic domain of the transglycosylase Slt70 from Escherichia coli and to lysozymes. The gp144 catalytic domain has an elongated groove that can bind at least five sugar residues at sites A-E. As in other lysozymes, the peptidoglycan cleavage (catalyzed by Glu 115 in gp144) occurs between sugar-binding subsites D and E. The x-ray structure of the phi KZ transglycosylase complexed with the chitotetraose (N-acetylglucosamine)(4) has been determined to 2.6-A resolution. The N-acetylglucosamine residues of the chitotetraose bind in sites A-D.     

The guanidino-group modifying enzymes: structural basis for their diversity and commonality

The guanidino-group modifying enzyme (GME) superfamily contains many drug targets, including metabolic enzymes from pathogenic microorganisms as well as key regulatory proteins from higher eukaryotes. These enzymes, despite their diverse sequences, adopt the common alpha/beta propeller fold and catalyze the modification of (methylated) guanidino groups. Our structural superposition and structure-based alignment for the GMEs have identified key residues that are involved in the catalysis and substrate binding. We have shown that conserved guanidino-carboxyl interactions are utilized in two different ways; the acidic residues in the catalytic site form hydrogen bonds to the substrate guanidino group, and the enzyme Arg residues at several key positions recognize the carboxyl group of the substrate and fix its orientation. Based on this observation, we have proposed rules for classifying the GME sequences and predicting their molecular function from the conservation of the key acidic and Arg residues. Other novel motifs have been identified, which involve residues that are not in direct contact with the substrate but are likely to stabilize the active-site conformation through hydrogen-bonding networks. In addition, we have examined the domain architecture of the GMEs. Although most members consist of a single catalytic domain, fold recognition analysis has identified a likely bifunctional enzyme from a cyanobacterium. It has also revealed common immunoglobulin-like beta-sandwich domains found in the enzymes that recognize protein substrates. These findings will be useful for predicting the precise mechanism of action for potential novel targets and designing therapeutic compounds against them.     

The structure of the RlmB 23S rRNA methyltransferase reveals a new methyltransferase fold with a unique knot

In Escherichia coli, RlmB catalyzes the methylation of guanosine 2251, a modification conserved in the peptidyltransferase domain of 23S rRNA. The crystal structure of this 2'O-methyltransferase has been determined at 2.5 A resolution. RlmB consists of an N-terminal domain connected by a flexible extended linker to a catalytic C-terminal domain and forms a dimer in solution. The C-terminal domain displays a divergent methyltransferase fold with a unique knotted region, and lacks the classic AdoMet binding site features. The N-terminal domain is similar to ribosomal proteins L7 and L30, suggesting a role in 23S rRNA recognition. The conserved residues in this novel family of 2'O-methyltransferases cluster in the knotted region, suggesting the location of the catalytic and AdoMet binding sites.     

The mdoC Gene of Escherichia coli Encodes a Membrane Protein That Is Required for Succinylation of Osmoregulated Periplasmic Glucans

Osmoregulated periplasmic glucans (OPGs) of Escherichia coli are anionic oligosaccharides that accumulate in the periplasmic space in response to low osmolarity of the medium. Their anionic character is provided by the substitution of the glucosidic backbone by phosphoglycerol originating from the membrane phospholipids and by succinyl residues from unknown origin. A phosphoglycerol-transferase-deficient mdoB mutant was subjected to Tn5 transposon mutagenesis, and putative mutant clones were screened for changes in the anionic character of OPGs by thin-layer chromatography. One mutant deficient in succinylation of OPGs was obtained, and the gene inactivated in this mutant was characterized and named mdoC. mdoC, which encodes a membrane-bound protein, is closely linked to the mdoGH operon necessary for the synthesis of the OPG backbone.     

Crystal structure of human carboxypeptidase M, a membrane-bound enzyme that regulates peptide hormone activity

Carboxypeptidase M (CPM), an extracellular glycosylphosphatidyl-inositol(GPI)-anchored membrane glycoprotein belonging to the CPN/E subfamily of "regulatory" metallo-carboxypeptidases, specifically removes C-terminal basic residues from peptides and proteins. Due to its wide distribution in human tissues, CPM is believed to play important roles in the control of peptide hormone and growth factor activity at the cell surface, and in the membrane-localized degradation of extracellular proteins. We have crystallized human GPI-free CPM, and have determined and refined its 3.0A crystal structure. The structure analysis reveals that CPM consists of a 295 residue N-terminal catalytic domain similar to that of duck CPD-2 (but only distantly related to CPA/B), an adjacent 86 residue beta-sandwich C-terminal domain characteristic of the CPN/E family but more conically shaped than the equivalent domain in CPD-2, and a unique, partially disordered 25 residue C-terminal extension to which the GPI membrane-anchor is post-translationally attached. Through this GPI anchor, and presumably via some positively charged side-chains of the C-terminal domain, the CPM molecule may interact with the membrane in such a way that its active centre will face alongside, i.e. well suited to interact with other membrane-bound protein substrates or small peptides. Modelling of the C-terminal part of the natural substrate Arg(6)-Met-enkephalin into the active site shows that the S1' pocket of CPM is particularly well designed to accommodate P1'-Arg residues, in agreement with the preference of CPM for cleaving C-terminal Arg.     

Osmoregulated periplasmic glucans of the free-living photosynthetic bacterium Rhodobacter sphaeroides.

The osmoregulated periplasmic glucans (OPGs) produced by Rhodobacter sphaeroides, a free-living organism, were isolated by trichloracetic acid treatment and gel permeation chromatography. Compounds obtained were characterized by compositional analysis, matrix-assisted laser desorption ionization mass spectrometry and nuclear magnetic resonance. R. sphaeroides predominantly synthesizes a cyclic glucan containing 18 glucose residues that can be substituted by one to seven succinyl esters residues at the C6 position of some of the glucose residues, and by one or two acetyl residues. The glucans were subjected to a mild alkaline treatment in order to remove the succinyl and acetyl substituents, analyzed by MALDI mass spectrometry and purified by high-performance anion-exchange chromatography. Methylation analysis revealed that this glucan is linked by 17 1,2 glycosidic bonds and one 1,6 glycosidic bond. Homonuclear and (1)H/(13)C heteronuclear NMR experiments revealed the presence of a single alpha-1,6 glycosidic linkage, whereas all other glucose residues are beta-1,2 linked. The different anomeric proton signals allowed a complete sequence-specific assignment of the glucan. The structural characteristics of this glucan are very similar to the previously described OPGs of Ralstonia solanacearum and Xanthomonas campestris, except for its different size and the presence of substituents. Therefore, similar OPGs are synthesized by phytopathogenic as well as free-living bacteria, suggesting these compounds are intrinsic components of the Gram-negative bacterial envelope.     

The crystal structure of Thermotoga maritima maltosyltransferase and its implications for the molecular basis of the novel transfer specificity.

Maltosyltransferase (MTase) from the hyperthermophile Thermotoga maritima represents a novel maltodextrin glycosyltransferase acting on starch and malto-oligosaccharides. It catalyzes the transfer of maltosyl units from alpha-1,4-linked glucans or malto-oligosaccharides to other alpha-1,4-linked glucans, malto-oligosaccharides or glucose. It belongs to the glycoside hydrolase family 13, which represents a large group of (beta/alpha)(8) barrel proteins sharing a similar active site structure. The crystal structures of MTase and its complex with maltose have been determined at 2.4 A and 2.1 A resolution, respectively. MTase is a homodimer, each subunit of which consists of four domains, two of which are structurally homologous to those of other family 13 enzymes. The catalytic core domain has the (beta/alpha)(8) barrel fold with the active-site cleft formed at the C-terminal end of the barrel. Substrate binding experiments have led to the location of two distinct maltose-binding sites; one lies in the active-site cleft, covering subsites -2 and -1; the other is located in a pocket adjacent to the active-site cleft. The structure of MTase, together with the conservation of active-site residues among family 13 glycoside hydrolases, are consistent with a common double-displacement catalytic mechanism for this enzyme. Analysis of maltose binding in the active site reveals that the transfer of dextrinyl residues longer than a maltosyl unit is prevented by termination of the active-site cleft after the -2 subsite by the side-chain of Lys151 and the stretch of residues 314-317, providing an explanation for the strict transfer specificity of MTase.     

Improving enzyme characteristics by gene shuffling; application to β-glucosidase

The genes of family 3 β-glucosidase enzymes consist of five distinct regions; the N-terminal residues, an N-terminal catalytic domain, a nonhomologous region, a C-terminal domain of unknown function and the C-terminal residues. The β-glucosidase genes derived from Cellvibrio gilvus (CG) and Agrobacterium tumefaciens (AT) have been subjected to gene deletion, truncation and shuffling. The folding information was found to be distributed unevenly across the different regions based on the gene manipulation results. Chimeric enzymes with improved enzyme characteristics were obtained only by gene shuffling at the C-terminal domain.     

Molecular characterization and expression in Escherichia coli of three beta-1,3-glucanase genes from Lysobacter enzymogenes strain N4-7

Lysobacter enzymogenes strain N4-7 produces multiple biochemically distinct extracellular beta-1,3-glucanase activities. The gluA, gluB, and gluC genes, encoding enzymes with beta-1,3-glucanase activity, were identified by a reverse-genetics approach following internal amino acid sequence determination of beta-1,3-glucanase-active proteins partially purified from culture filtrates of strain N4-7. Analysis of gluA and gluC gene products indicates that they are members of family 16 glycoside hydrolases that have significant sequence identity to each other throughout the catalytic domain but that differ structurally by the presence of a family 6 carbohydrate-binding domain within the gluC product. Analysis of the gluB gene product indicates that it is a member of family 64 glycoside hydrolases. Expression of each gene in Escherichia coli resulted in the production of proteins with beta-1,3-glucanase activity. Biochemical analyses of the recombinant enzymes indicate that GluA and GluC exhibit maximal activity at pH 4.5 and 45 degrees C and that GluB is most active between pH 4.5 and 5.0 at 41 degrees C. Activity of recombinant proteins against various beta-1,3 glucan substrates indicates that GluA and GluC are most active against linear beta-1,3 glucans, while GluB is most active against the insoluble beta-1,3 glucan substrate zymosan A. These data suggest that the contribution of beta-1,3-glucanases to the biocontrol activity of L. enzymogenes may be due to complementary activities of these enzymes in the hydrolysis of beta-1,3 glucans from fungal cell walls.     

The solution structure of a domain from the Neisseria meningitidis lipoprotein PilP reveals a new beta-sandwich fold

Type IV pili are long, thin fibres, which extend from the surface of the bacterial pathogen Neisseria meningitidis; they play a key role in adhesion and colonisation of host cells. PilP is a lipoprotein, suggested to be involved in the assembly and stabilization of an outer membrane protein, PilQ, which is required for pilus formation. Here we describe the expression of a recombinant fragment of PilP, spanning residues 20 to 181, and determination of the solution structure of a folded domain, spanning residues 85 to 163, by NMR. The N-terminal third of the protein, from residues 20 to 84, is apparently unfolded. Protease digestion yielded a 113 residue fragment that contained the folded domain. The domain adopts a simple beta-sandwich type fold, consisting of a three-stranded beta-sheet packed against a four-stranded beta-sheet. There is also a short segment of 3(10) helix at the N-terminal part of the folded domain. We were unable to identify any other proteins that are closely related in structure to the PilP domain, although the fold appears to be distantly related to the lipocalin family. Over 40 homologues of PilP have been identified in Gram-negative bacteria and the majority of conserved residues lie within the folded domain. The fourth beta-strand and adjacent loop regions contain a high proportion of conserved residues, including three glycine residues, which seem to play a role in linking the two beta-sheets. The two beta-sheets pack together to form a crevice, lined with conserved hydrophobic residues: we suggest that this feature could act as a binding site for a small ligand. The results show that PilP and its homologues have a conserved, folded domain at the C-terminal end of the protein that may be involved in mediating binding to hydrophobic ligands.     

Bacterial 1,3-1,4-beta-glucanases: structure, function and protein engineering

1,3-1,4-beta-Glucanases (or lichenases, EC 3.2.1.73) hydrolyse linear beta-glucans containing beta-1,3 and beta-1,4 linkages such as cereal beta-glucans and lichenan, with a strict cleavage specificity for beta-1,4 glycosidic bonds on 3-O-substituted glucosyl residues. The bacterial enzymes are retaining glycosyl hydrolases of family 16 with a jellyroll beta-sandwich fold and a substrate binding cleft composed of six subsites. The present paper reviews the structure-function aspects of the enzymatic action including mechanistic enzymology, protein engineering and X-ray crystallographic studies.     

The crystal structure of leucyl/phenylalanyl-tRNA-protein transferase from Escherichia coli

Leucyl/phenylalanyl-tRNA-protein transferase (L/F-transferase) is an N-end rule pathway enzyme, which catalyzes the transfer of Leu and Phe from aminoacyl-tRNAs to exposed N-terminal Arg or Lys residues of acceptor proteins. Here, we report the 1.6 A resolution crystal structure of L/F-transferase (JW0868) from Escherichia coli, the first three-dimensional structure of an L/F-transferase. The L/F-transferase adopts a monomeric structure consisting of two domains that form a bilobate molecule. The N-terminal domain forms a small lobe with a novel fold. The large C-terminal domain has a highly conserved fold, which is observed in the GCN5-related N-acetyltransferase (GNAT) family. Most of the conserved residues of L/F-transferase reside in the central cavity, which exists at the interface between the N-terminal and C-terminal domains. A comparison of the structures of L/F-transferase and the bacterial peptidoglycan synthase FemX, indicated a structural homology in the C-terminal domain, and a similar domain interface region. Although the peptidyltransferase function is shared between the two proteins, the enzymatic mechanism would differ. The conserved residues in the central cavity of L/F-transferase suggest that this region is important for the enzyme catalysis.     

B. subtilis ykuD protein at 2.0 A resolution: insights into the structure and function of a novel, ubiquitous family of bacterial enzymes

The crystal structure of the product of the Bacillus subtilis ykuD gene was solved by the multiwavelength anomalous dispersion (MAD) method and refined using data to 2.0 A resolution. The ykuD protein is a representative of a distinctly prokaryotic and ubiquitous family found among both pathogenic and nonpathogenic Gram-positive and Gram-negative bacteria. The deduced amino acid sequence reveals the presence of an N-terminal LysM domain, which occurs among enzymes involved in cell wall metabolism, and a novel, putative catalytic domain with a highly conserved His/Cys-containing motif of hitherto unknown structure. As the wild-type protein did not crystallize, a double mutant was designed (Lys117Ala/Gln118Ala) to reduce excess surface conformational entropy. As expected, the structure of the LysM domain is similar to the NMR structure reported for an analogous domain from Escherichia coli murein transglycosylase MltD. The molecular model also shows that the 112-residue-long C-terminal domain has a novel tertiary fold consisting of a beta-sandwich with two mixed sheets, one containing five strands and the other, six strands. The two beta-sheets form a cradle capped by an alpha-helix. This domain contains a putative catalytic site with a tetrad of invariant His123, Gly124, Cys139, and Arg141. The stereochemistry of this active site shows similarities to peptidotransferases and sortases, and suggests that the enzymes of the ykuD family may play an important role in cell wall biology.     

The N-terminal domain of the regulatory subunit is sufficient for complete activation of acetohydroxyacid synthase III from Escherichia coli

We have previously proposed a model for the fold of the N-terminal domain of the small, regulatory subunit (SSU) of acetohydroxyacid synthase isozyme III. The fold is an alpha-beta sandwich with betaalphabetabetaalphabeta topology, structurally homologous to the C-terminal regulatory domain of 3-phosphoglycerate dehydrogenase. We suggested that the N-terminal domains of a pair of SSUs interact in the holoenzyme to form two binding sites for the feedback inhibitor valine in the interface between them. The model was supported by mutational analysis and other evidence. We have now examined the role of the C-terminal portion of the SSU by construction of truncated polypeptides (lacking 35, 48, 80, 95, or 112 amino acid residues from the C terminus) and examining the properties of holoenzymes reconstituted using these constructs. The Delta35, Delta48, and Delta80 constructs all lead to essentially complete activation of the catalytic subunits. The Delta80 construct, corresponding to the putative N-terminal domain, has the highest level of affinity for the catalytic subunits and leads to a reconstituted enzyme with k(cat)/K(M) about twice that of the wild-type enzyme. On the other hand, none of these constructs binds valine or leads to a valine-sensitive enzyme on reconstitution. The enzyme reconstituted with the Delta80 construct does not bind valine, either. The N-terminal portion (about 80 amino acid residues) of the SSU is thus necessary and sufficient for recognition and activation of the catalytic subunits, but the C-terminal half of the SSU is required for valine binding and response. We suggest that the C-terminal region of the SSU contributes to monomer-monomer interactions, and provide additional experimental evidence for this suggestion.