The structure of the exopolyphosphatase (PPX) from Escherichia coli O157:H7 suggests a binding mode for long polyphosphate chains

Polyphosphate (polyP) is a linear polymer consisting of tens to hundreds of phosphate molecules joined together by high-energy anhydride bonds. These polymers are found in virtually all prokaryotic and eukaryotic cells and perform many functions; prominent among them are the responses to many stresses. Polyphosphate is synthesized by polyP kinase (PPK), using the terminal phosphate of ATP as the substrate, and degraded to inorganic phosphate by both endo- and exopolyphosphatases. Here we report the crystal structure and analysis of the polyphosphate phosphatase PPX from Escherichia coli O157:H7 refined at 2.2 Angstroms resolution. PPX is made of four domains. Domains I and II display structural similarity with one another and share the ribonuclease-H-like fold. Domain III bears structural similarity to the N-terminal, HD domain of SpoT. Domain IV, the smallest domain, has structural counterparts in cold-shock associated RNA-binding proteins but is of unknown function in PPX. The putative PPX active site is located at the interface between domains I and II. In the crystal structure of PPX these two domains are close together and represent the "closed" state. Comparison with the crystal structure of PPX/GPPA from Aquifex aeolicus reveals close structural similarity between domains I and II of the two enzymes, with the PPX/GPPA representing an "open" state. A striking feature of the dimer is a deep S-shaped canyon extending along the dimer interface and lined with positively charged residues. The active site region opens to this canyon. We postulate that this is a likely site of polyP binding.     

The crystal structure of a novel, latent dihydroorotase from Aquifex aeolicus at 1.7A resolution

Dihydroorotases (EC 3.5.2.3) catalyze the reversible cyclization of carbamoyl aspartate to form dihydroorotate in de novo pyrimidine biosynthesis. The X-ray structures of Aquifex aeolicus dihydroorotase in two space groups, C222(1) and C2, were determined at a resolution of 1.7A. These are the first structures of a type I dihydroorotase, a class of molecules that includes the dihydroorotase domain of mammalian CAD. The type I enzymes are more ancient and larger, at 45 kDa, than the type II enzymes exemplified by the 38 kDa Escherichia coli dihydroorotase. Both dihydroorotases are members of the metallo-dependent hydrolase superfamily, whose members have a distorted "TIM barrel" domain containing the active site. However, A.aeolicus dihydroorotase has a second, composite domain, which the E.coli enzyme lacks and has only one of the two zinc atoms present in the E.coli enzyme. A.aeolicus dihydroorotase is unique in exhibiting significant activity only when complexed with aspartate transcarbamoylase, whereas the E.coli dihydroorotase and the CAD dihydroorotase domain are active as free proteins. The latency of A.aeolicus dihydroorotase can be related to two differences between its structure and that of E.coli dihydroorotase: (1) the monoclinic structure has a novel cysteine ligand to the zinc that blocks the active site and possibly functions as a "cysteine switch"; and (2) active site residues that bind the substrate in E.coli dihydroorotase are located in disordered loops in both crystal structures of A.aeolicus dihydroorotase and may function as a disorder-to-order "entropy switch".     

The structure of 3-methylaspartase from Clostridium tetanomorphum functions via the common enolase chemical step.

Methylaspartate ammonia-lyase (3-methylaspartase, MAL; EC ) catalyzes the reversible anti elimination of ammonia from L-threo-(2S,3S)-3-methylaspartic acid to give mesaconic acid. This reaction lies on the main catabolic pathway for glutamate in Clostridium tetanomorphum. MAL requires monovalent and divalent cation cofactors for full catalytic activity. The enzyme has attracted interest because of its potential use as a biocatalyst. The structure of C. tetanomorphum MAL has been solved to 1.9-A resolution by the single-wavelength anomalous diffraction method. A divalent metal ion complex of the protein has also been determined. MAL is a homodimer with each monomer consisting of two domains. One is an alpha/beta-barrel, and the other smaller domain is mainly beta-strands. The smaller domain partially occludes the C terminus of the barrel and forms a large cleft. The structure identifies MAL as belonging to the enolase superfamily of enzymes. The metal ion site is located in a large cleft between the domains. Potential active site residues have been identified based on a combination of their proximity to a metal ion site, molecular modeling, and sequence homology. In common with all members of the enolase superfamily, the carboxylic acid of the substrate is co-ordinated by the metal ions, and a proton adjacent to a carboxylic acid group of the substrate is abstracted by a base. In MAL, it appears that Lys(331) removes the alpha-proton of methylaspartic acid. This motif is the defining mechanistic characteristic of the enolase superfamily of which all have a common fold. The degree of structural conservation is remarkable given only four residues are absolutely conserved.     

Aspergillus aculeatus beta-1,4-galactanase: substrate recognition and relations to other glycoside hydrolases in clan GH-A

The three-dimensional structure of Aspergillus aculeatus beta-1,4-galactanase (AAGAL), an enzyme involved in pectin degradation, has been determined by multiple isomorphous replacement to 2.3 and 1.8 A resolution at 293 and 100 K, respectively. It represents the first known structure for a polysaccharidase with this specificity and for a member of glycoside hydrolase family 53 (GH-53). The enzyme folds into a (beta/alpha)(8) barrel with the active site cleft located at the C-terminal side of the barrel consistent with the classification of GH-53 in clan GH-A, a superfamily of enzymes with common fold and catalytic machinery but diverse specificities. Putative substrate-enzyme interactions were elucidated by modeling of beta-1,4-linked galactobioses into the possible substrate binding subsites. The structure and modeling studies identified five potential subsites for the binding of galactans, of which one is a pocket suited for accommodating the arabinan side chain in arabinogalactan, one of the natural substrates. A comparison with the substrate binding grooves of other Clan GH-A enzymes suggests that shape complementarity is crucial in determining the specificity of polysaccharidases.     

Structural comparisons of TIM barrel proteins suggest functional and evolutionary relationships between beta-galactosidase and other glycohydrolases

Beta-galactosidase (lacZ) from Escherichia coli is a 464 kDa homotetramer. Each subunit consists of five domains, the third being an alpha/beta barrel that contains most of the active site residues. A comparison is made between each of the domains and a large set of proteins representative of all structures from the protein data bank. Many structures include an alpha/beta barrel. Those that are most similar to the alpha/beta barrel of E. coli beta-galactosidase have similar catalytic residues and belong to the so-called "4/7 superfamily" of glycosyl hydrolases. The structure comparison suggests that beta-amylase should also be included in this family. Of three structure comparison methods tested, the "ProSup" procedure of Zu-Kang and Sippl and the "Superimpose" procedure of Diederichs were slightly superior in discriminating the members of this superfamily, although all procedures were very powerful in identifying related protein structures. Domains 1, 2, and 4 of E. coli beta-galactosidase have topologies related to "jelly-roll barrels" and "immunoglobulin constant" domains. This fold also occurs in the cellulose binding domains (CBDs) of a number of glycosyl hydrolases. The fold of domain 1 of E. coli beta-galactosidase is closely related to some CBDs, and the domain contributes to substrate binding, but in a manner unrelated to cellulose binding by the CBDs. This is typical of domains 1, 2, 4, and 5, which appear to have been recruited to play roles in beta-galactosidase that are unrelated to the functions that such domains provide in other contexts. It is proposed that beta-galactosidase arose from a prototypical single domain alpha/beta barrel with an extended active site cleft. The subsequent incorporation of elements from other domains could then have reduced the size of the active site from a cleft to a pocket to better hydrolyze the disaccharide lactose and, at the same time, to facilitate the production of inducer, allolactose.     

Identification of a PD-(D/E)XK-like domain with a novel configuration of the endonuclease active site in the methyl-directed restriction enzyme Mrr and its homologs

The Escherichia coli K-12 restriction enzyme Mrr recognizes and cleaves N6-methyladenine- and 5-methylcytosine-containing DNA. Its amino acid sequence has been subjected to structure prediction and comparison with other sequences from publicly available sources. The results obtained suggest that Mrr and related putative endonucleases possess a cleavage domain typical for all the so far structurally characterized type II restriction enzymes, however with an unusual glutamine residue at the central position of the (D/E)-(D/E)XK hallmark of the active site. The "missing" acidic side chain was instead found anchored in a different, unusual position, suggesting that Mrr represents a third topological variant of the endonuclease active site in addition to the two alternatives determined previously (Skirgaila et al., 1998. J. Mol. Biol. 279, 473-481). One of the newly identified putative endonucleases from the Mrr family is composed of two diverged cleavage domains, which possess both the "typical" D-EXK and the "Mrr-like" D-QXK variants of the active site. Among the Mrr homologs there are also proteins from yeast, in which restriction phenotype has not been observed, suggesting that the free-standing Eukaryotic PD-(D/E)XK superfamily members might be implicated in other cellular processes involving enzymatic DNA cleavage.     

Crystal structure of an inactive duck delta II crystallin mutant with bound argininosuccinate

Delta-crystallin, the major soluble protein component of avian and reptilian eye lenses, is highly homologous to the urea cycle enzyme, argininosuccinate lyase (ASL). In duck lenses, there are two highly homologous delta crystallins, delta I and delta II, that are 94% identical in amino acid sequence. While delta II crystallin has been shown to exhibit ASL activity in vitro, delta I is enzymatically inactive. The X-ray structure of a His to Asn mutant of duck delta II crystallin (H162N) with bound argininosuccinate has been determined to 2.3 A resolution using the molecular replacement technique. The overall fold of the protein is similar to other members of the superfamily to which this protein belongs, with the active site located in a cleft formed by three different monomers in the tetramer. The active site of the H162N mutant structure reveals that the side chain of Glu 296 has a different orientation relative to the homologous residue in the H91N mutant structure [Abu-Abed et al. (1997) Biochemistry 36, 14012-14022]. This shift results in the loss of the hydrogen bond between His 162 and Glu 296 seen in the H91N and turkey delta I crystallin structures; this H-bond is believed to be crucial for the catalytic mechanism of ASL/delta II crystallin. Argininosuccinate was found to be bound to residues in each of the three monomers that form the active site. The fumarate moiety is oriented toward active site residues His 162 and Glu 296 and other residues that are part of two of the three highly conserved regions of amino acid sequence in the superfamily, while the arginine moiety of the substrate is oriented toward residues which belong to either domain 1 or domain 2. The analysis of the structure reveals that significant conformational changes occur on substrate binding. The comparison of this structure with the inactive turkey delta I crystallin reveals that the conformation of domain 1 is crucial for substrate affinity and that the delta I protein is almost certainly inactive because it can no longer bind the substrate.     

Crystal structure of the Holliday junction resolving enzyme T7 endonuclease I

We have solved the crystal structure of the Holliday junction resolving enzyme T7 endonuclease I at 2.1 A resolution using the multiwavelength anomalous dispersion (MAD) technique. Endonuclease I exhibits strong structural specificity for four-way DNA junctions. The structure shows that it forms a symmetric homodimer arranged in two well-separated domains. Each domain, however, is composed of elements from both subunits, and amino acid side chains from both protomers contribute to the active site. While no significant structural similarity could be detected with any other junction resolving enzyme, the active site is similar to that found in several restriction endonucleases. T7 endonuclease I therefore represents the first crystal structure of a junction resolving enzyme that is a member of the nuclease superfamily of enzymes.     

Crystal structure of a type III pantothenate kinase: insight into the mechanism of an essential coenzyme A biosynthetic enzyme universally distributed in bacteria

Pantothenate kinase (PanK) catalyzes the first step in the five-step universal pathway of coenzyme A (CoA) biosynthesis, a key transformation that generally also regulates the intracellular concentration of CoA through feedback inhibition. A novel PanK protein encoded by the gene coaX was recently identified that is distinct from the previously characterized type I PanK (exemplified by the Escherichia coli coaA-encoded PanK protein) and type II eukaryotic PanKs and is not inhibited by CoA or its thioesters. This type III PanK, or PanK-III, is widely distributed in the bacterial kingdom and accounts for the only known PanK in many pathogenic species, such as Helicobacter pylori, Bordetella pertussis, and Pseudomonas aeruginosa. Here we report the first crystal structure of a type III PanK, the enzyme from Thermotoga maritima (PanK(Tm)), solved at 2.0-A resolution. The structure of PanK(Tm) reveals that type III PanKs belong to the acetate and sugar kinase/heat shock protein 70/actin (ASKHA) protein superfamily and that they retain the highly conserved active site motifs common to all members of this superfamily. Comparative structural analysis of the PanK(Tm) active site configuration and mutagenesis of three highly conserved active site aspartates identify these residues as critical for PanK-III catalysis. Furthermore, the analysis also provides an explanation for the lack of CoA feedback inhibition by the enzyme. Since PanK-III adopts a different structural fold from that of the E. coli PanK -- which is a member of the "P-loop kinase"superfamily -- this finding represents yet another example of convergent evolution of the same biological function from a different protein ancestor.     

Crystal structure of muconate lactonizing enzyme at 3 A resolution

The crystal structure of muconate lactonizing enzyme has been solved at 3 A resolution, and an unambiguous alpha-carbon backbone chain trace made. The enzyme contains three domains; the central domain is a parallel-stranded alpha-beta barrel, which has previously been reported in six other enzymes, including triose phosphate isomerase and pyruvate kinase. One novel feature of this enzyme is that its alpha-beta barrel has only seven parallel alpha-helices around the central core of eight parallel beta-strands; all other known alpha-beta barrels contain eight such helices. The N-terminal (alpha + beta) and C-terminal domains cover the cleft where the eighth helix would be. The active site of muconate lactonizing enzyme has been found by locating the manganese ion that is essential for catalytic activity, and by binding and locating an inhibitor, alpha-ketoglutarate. The active site lies in a cleft between the N-terminal and barrel domains; when the active sites of muconate lactonizing enzyme and triose phosphate isomerase are superimposed, barrel-strand 1 of triose phosphate isomerase is aligned with barrel-strand 3 of muconate lactonizing enzyme. This implies that structurally homologous active-site residues in the two enzymes are carried on different parts of the primary sequence; the ancestral gene would had to have been transposed during its evolution to the modern proteins, which seems unlikely. Therefore, these two enzymes may be related by convergent, rather than divergent, evolution.     

X-ray crystal structure of the hypothetical phosphotyrosine phosphatase MDP-1 of the haloacid dehalogenase superfamily

The haloacid dehalogenase (HAD) superfamily is comprised of structurally homologous enzymes that share several conserved sequence motifs (loops I-IV) in their active site. The majority of HAD members are phosphohydrolases and may be divided into three subclasses depending on domain organization. In classes I and II, a mobile "cap" domain reorients upon substrate binding, closing the active site to bulk solvent. Members of the third class lack this additional domain. Herein, we report the 1.9 A X-ray crystal structures of a member of the third subclass, magnesium-dependent phosphatase-1 (MDP-1) both in its unliganded form and with the product analogue, tungstate, bound to the active site. The secondary structure of MDP-1 is similar to that of the "core" domain of other type I and type II HAD members with the addition of a small, 28-amino acid insert that does not close down to exclude bulk solvent in the presence of ligand. In addition, the monomeric oligomeric state of MDP-1 does not allow the participation of a second subunit in the formation and solvent protection of the active site. The binding sites for the phosphate portion of the substrate and Mg(II) cofactor are also similar to those of other HAD members, with all previously observed contacts conserved. Unlike other subclass III HAD members, MDP-1 appears to be equally able to dephosphorylate phosphotyrosine and closed-ring phosphosugars. Modeling of possible substrates in the active site of MDP-1 reveals very few potential interactions with the substrate leaving group. The mapping of conserved residues in sequences of MDP-1 from different eukaryotic organisms reveals that they colocalize to a large region on the surface of the protein outside the active site. This observation combined with the modeling studies suggests that the target of MDP-1 is most likely a phosphotyrosine in an unknown protein rather than a small sugar-based substrate.     

The crystal structure of anthranilate phosphoribosyltransferase from the enterobacterium Pectobacterium carotovorum

The structure of anthranilate phosphoribosyltransferase from the enterobacterium Pectobacterium carotovorum has been solved at 2.4 A in complex with Mn(2+)-pyrophosphate, and at 1.9 A without ligands. The enzyme structure has a novel phosphoribosyltransferase (PRT) fold and displays close homology to the structures of pyrimidine nucleoside phosphorylases. The enzyme is a homodimer with a monomer of 345 residues. Each monomer consists of two subdomains, alpha and alpha/beta, which form a cleft containing the active site. The nature of the active site is inferred from the trapped MnPPi complex and detailed knowledge of the active sites of nucleoside phosphorylases. With the anthranilate (An)PRT structure solved, the structures of all the enzymes required for tryptophan biosynthesis are now known.     

Crystal structure of Anaerobiospirillum succiniciproducens PEP carboxykinase reveals an important active site loop

The 2.2 Angstroms resolution crystal structure of the enzyme phosphoenolpyruvate carboxykinase (PCK) from the bacterium Anaerobiospirillum succiniciproducens complexed with ATP, Mg(2+), Mn(2+) and the transition state analogue oxalate has been solved. The 2.4 Angstroms resolution native structure of A. succiniciproducens PCK has also been determined. It has been found that upon binding of substrate, PCK undergoes a conformational change. Two domains of the molecule fold towards each other, with the substrates and metal ions held in a cleft formed between the two domains. This domain movement is believed to accelerate the reaction PCK catalyzes by forcing bulk solvent molecules out of the active site. Although the crystal structure of A. succiniciproducens PCK with bound substrate and metal ions is related to the structures of PCK from Escherichia coli and Trypanosoma cruzi, it is the first crystal structure from this class of enzymes that clearly shows an important surface loop (residues 383-397) from the C-terminal domain, hydrogen bonding with the peptide backbone of the active site residue Arg60. The interaction between the surface loop and the active site backbone, which is a parallel beta-sheet, seems to be a feature unique of A. succiniciproducens PCK. The association between the loop and the active site is the third type of interaction found in PCK that is thought to play a part in the domain closure. This loop also appears to help accelerate catalysis by functioning as a 'lid' that shields water molecules from the active site.     

Crystal structure of quinohemoprotein alcohol dehydrogenase from Comamonas testosteroni: structural basis for substrate oxidation and electron transfer.

Quinoprotein alcohol dehydrogenases are redox enzymes that participate in distinctive catabolic pathways that enable bacteria to grow on various alcohols as the sole source of carbon and energy. The x-ray structure of the quinohemoprotein alcohol dehydrogenase from Comamonas testosteroni has been determined at 1.44 A resolution. It comprises two domains. The N-terminal domain has a beta-propeller fold and binds one pyrroloquinoline quinone cofactor and one calcium ion in the active site. A tetrahydrofuran-2-carboxylic acid molecule is present in the substrate-binding cleft. The position of this oxidation product provides valuable information on the amino acid residues involved in the reaction mechanism and their function. The C-terminal domain is an alpha-helical type I cytochrome c with His(608) and Met(647) as heme-iron ligands. This is the first reported structure of an electron transfer system between a quinoprotein alcohol dehydrogenase and cytochrome c. The shortest distance between pyrroloquinoline quinone and heme c is 12.9 A, one of the longest physiological edge-to-edge distances yet determined between two redox centers. A highly unusual disulfide bond between two adjacent cysteines bridges the redox centers. It appears essential for electron transfer. A water channel delineates a possible pathway for proton transfer from the active site to the solvent.     

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.     

Three-dimensional structure of Erwinia chrysanthemi pectin methylesterase reveals a novel esterase active site

Most structures of neutral lipases and esterases have been found to adopt the common alpha/beta hydrolase fold and contain a catalytic Ser-His-Asp triad. Some variation occurs in both the overall protein fold and in the location of the catalytic triad, and in some enzymes the role of the aspartate residue is replaced by a main-chain carbonyl oxygen atom. Here, we report the crystal structure of pectin methylesterase that has neither the common alpha/beta hydrolase fold nor the common catalytic triad. The structure of the Erwinia chrysanthemi enzyme was solved by multiple isomorphous replacement and refined at 2.4 A to a conventional crystallographic R-factor of 17.9 % (R(free) 21.1 %). This is the first structure of a pectin methylesterase and reveals the enzyme to comprise a right-handed parallel beta-helix as seen in the pectinolytic enzymes pectate lyase, pectin lyase, polygalacturonase and rhamnogalacturonase, and unlike the alpha/beta hydrolase fold of rhamnogalacturonan acetylesterase with which it shares esterase activity. Pectin methylesterase has no significant sequence similarity with any protein of known structure. Sequence conservation among the pectin methylesterases has been mapped onto the structure and reveals that the active site comprises two aspartate residues and an arginine residue. These proposed catalytic residues, located on the solvent-accessible surface of the parallel beta-helix and in a cleft formed by external loops, are at a location similar to that of the active site and substrate-binding cleft of pectate lyase. The structure of pectin methylesterase is an example of a new family of esterases.     

Crystal structure of the PdxY Protein from Escherichia coli

The crystal structure of Escherichia coli PdxY, the protein product of the pdxY gene, has been determined to a 2.2-A resolution. PdxY is a member of the ribokinase superfamily of enzymes and has sequence homology with pyridoxal kinases that phosphorylate pyridoxal at the C-5' hydroxyl. The protein is a homodimer with an active site on each monomer composed of residues that come exclusively from each respective subunit. The active site is filled with a density that fits that of pyridoxal. In monomer A, the ligand appears to be covalently attached to Cys122 as a thiohemiacetal, while in monomer B it is not covalently attached but appears to be partially present as pyridoxal 5'-phosphate. The presence of pyridoxal phosphate and pyridoxal as ligands was confirmed by the activation of aposerine hydroxymethyltransferase after release of the ligand by the denaturation of PdxY. The ligand, which appears to be covalently attached to Cys122, does not dissociate after denaturation of the protein. A detailed comparison (of functional properties, sequence homology, active site and ATP-binding-site residues, and active site flap types) of PdxY with other pyridoxal kinases as well as the ribokinase superfamily in general suggested that PdxY is a member of a new subclass of the ribokinase superfamily. The structure of PdxY also permitted an interpretation of work that was previously published about this enzyme.     

Polyphosphate binding and chain length recognition of Escherichia coli exopolyphosphatase

Exopolyphosphatase of Escherichia coli (PPX) is a highly processive enzyme demonstrating the ability to recognize polyphosphates of specific lengths. The mechanisms responsible for the processivity and polymer length recognition of the enzyme were investigated in relation to the manner in which polyphosphate is bound to the enzyme. Multiple polyphosphate binding sites were identified on distant portions of the enzyme and were determined to be responsible for the polymer length recognition of the enzyme. In addition, two independently folded domains were identified. The N-terminal domain contained a quasi-processive polyphosphatase active site belonging to the sugar kinase/actin/hsp70 superfamily. The C-terminal domain contained a single polyphosphate binding site and was responsible for nearly all of the PPX affinity for polyphosphate. This domain was also found to confer a highly processive mode of action to PPX. Collectively, these results were used to describe the interaction of polyphosphate with PPX.     

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.     

Structural basis of severe acute respiratory syndrome coronavirus ADP-ribose-1''-phosphate dephosphorylation by a conserved domain of nsP3

The crystal structure of a conserved domain of nonstructural protein 3 (nsP3) from severe acute respiratory syndrome coronavirus (SARS-CoV) has been solved by single-wavelength anomalous dispersion to 1.4 A resolution. The structure of this "X" domain, seen in many single-stranded RNA viruses, reveals a three-layered alpha/beta/alpha core with a macro-H2A-like fold. The putative active site is a solvent-exposed cleft that is conserved in its three structural homologs, yeast Ymx7, Archeoglobus fulgidus AF1521, and Er58 from E. coli. Its sequence is similar to yeast YBR022W (also known as Poa1P), a known phosphatase that acts on ADP-ribose-1'-phosphate (Appr-1'-p). The SARS nsP3 domain readily removes the 1' phosphate group from Appr-1'-p in in vitro assays, confirming its phosphatase activity. Sequence and structure comparison of all known macro-H2A domains combined with available functional data suggests that proteins of this superfamily form an emerging group of nucleotide phosphatases that dephosphorylate Appr-1'-p.