RNA-catalyzed carbon-carbon bond formation.

RNA molecules with catalytic properties have been isolated by in vitro selection from combinatorial libraries. A broad range of chemical reactions can be catalyzed, and nucleic acids can accelerate bond formation between small organic substrates. The catalytic performance of nucleic acids can be enhanced by incorporation of additional functional groups. This minireview focuses on carbon-carbon bond formation accelerated by in vitro selected ribozymes.     

Crystal structure of the DsbB-DsbA complex reveals a mechanism of disulfide bond generation

Oxidation of cysteine pairs to disulfide requires cellular factors present in the bacterial periplasmic space. DsbB is an E. coli membrane protein that oxidizes DsbA, a periplasmic dithiol oxidase. To gain insight into disulfide bond formation, we determined the crystal structure of the DsbB-DsbA complex at 3.7 A resolution. The structure of DsbB revealed four transmembrane helices and one short horizontal helix juxtaposed with Cys130 in the mobile periplasmic loop. Whereas DsbB in the resting state contains a Cys104-Cys130 disulfide, Cys104 in the binary complex is engaged in the intermolecular disulfide bond and captured by the hydrophobic groove of DsbA, resulting in separation from Cys130. This cysteine relocation prevents the backward resolution of the complex and allows Cys130 to approach and activate the disulfide-generating reaction center composed of Cys41, Cys44, Arg48, and ubiquinone. We propose that DsbB is converted by its specific substrate, DsbA, to a superoxidizing enzyme, capable of oxidizing this extremely oxidizing oxidase.     

Crystal structure of S-adenosylhomocysteine hydrolase from rat liver.

The crystal structure of rat liver S-adenosyl-L-homocysteine hydrolase (AdoHcyase, EC 3.3.1.1) which catalyzes the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy) has been determined at 2.8 A resolution. AdoHcyase from rat liver is a tetrameric enzyme with 431 amino acid residues in each identical subunit. The subunit is composed of the catalytic domain, the NAD+-binding domain, and the small C-terminal domain. Both catalytic and NAD+-binding domains are folded into an ellipsoid with a typical alpha/beta twisted open sheet structure. The C-terminal section is far from the main body of the subunit and extends into the opposite subunit. An NAD+ molecule binds to the consensus NAD+-binding cleft of the NAD+-binding domain. The peptide folding pattern of the catalytic domain is quite similar to the patterns observed in many methyltransferases. Although the crystal structure does not contain AdoHcy or its analogue, there is a well-formed AdoHcy-binding crevice in the catalytic domain. Without introducing any major structural changes, an AdoHcy molecule can be placed in the catalytic domain. In the structure described here, the catalytic and NAD+-binding domains are quite far apart from each other. Thus, the enzyme appears to have an "open" conformation in the absence of substrate. It is likely that binding of AdoHcy induces a large conformational change so as to place the ribose moiety of AdoHcy in close proximity to the nicotinamide moiety of NAD+. A catalytic mechanism of AdoHcyase has been proposed on the basis of this crystal structure. Glu155 acts as a proton acceptor from the O3'-H when the proton of C3'-H is abstracted by NAD+. His54 or Asp130 acts as a general acid-base catalyst, while Cys194 modulates the oxidation state of the bound NAD+. The polypeptide folding pattern of the catalytic domain suggests that AdoHcy molecules can travel freely to and from AdoHcyase and methyltransferases to properly regulate methyltransferase activities. We believe that the crystal structure described here can provide insight into the molecular architecture of this important regulatory enzyme.     

The structure of the C-C bond hydrolase MhpC provides insights into its catalytic mechanism

2-Hydroxy-6-ketonona-2,4-diene-1,9-dioic acid 5,6-hydrolase (MhpC) is a 62 kDa homodimeric enzyme of the phenylpropionate degradation pathway of Escherichia coli. The 2.1 A resolution X-ray structure of the native enzyme determined from orthorhombic crystals confirms that it is a member of the alpha/beta hydrolase fold family, comprising eight beta-strands interconnected by loops and helices. The 2.8 A resolution structure of the enzyme co-crystallised with the non-hydrolysable substrate analogue 2,6-diketo-nona-1,9-dioic acid (DKNDA) confirms the location of the active site in a buried channel including Ser110, His263 and Asp235, postulated contributors to a serine protease-like catalytic triad in homologous enzymes. It appears that the ligand binds in two separate orientations. In the first, the C6 keto group of the inhibitor forms a hemi-ketal adduct with the Ser110 side-chain, the C9 carboxylate group interacts, via the intermediacy of a water molecule, with Arg188 at one end of the active site, while the C1 carboxylate group of the inhibitor comes close to His114 at the other end. In the second orientation, the C1 carboxylate group binds at the Arg188 end of the active site and the C9 carboxylate group at the His114 end. These arrangements implicated His114 or His263 as plausible contributors to catalysis of the initial enol/keto tautomerisation of the substrate but lack of conservation of His114 amongst related enzymes and mutagenesis results suggest that His263 is the residue involved. Variability in the quality of the electron density for the inhibitor amongst the eight molecules of the crystal asymmetric unit appears to correlate with alternative positions for the side-chain of His114. This might arise from half-site occupation of the dimeric enzyme and reflect the apparent dissociation of approximately 50% of the keto intermediate from the enzyme during the catalytic cycle.     

Crystal structure of a putative isochorismatase hydrolase from Oleispira antarctica

Isochorismatase-like hydrolases (IHL) constitute a large family of enzymes divided into five structural families (by SCOP). IHLs are crucial for siderophore-mediated ferric iron acquisition by cells. Knowledge of the structural characteristics of these molecules will enhance the understanding of the molecular basis of iron transport, and perhaps resolve which of the mechanisms previously proposed in the literature is the correct one. We determined the crystal structure of the apo-form of a putative isochorismatase hydrolase OaIHL (PDB code: 3LQY) from the antarctic γ-proteobacterium Oleispira antarctica, and did comparative sequential and structural analysis of its closest homologs. The characteristic features of all analyzed structures were identified and discussed. We also docked isochorismate to the determined crystal structure by in silico methods, to highlight the interactions of the active center with the substrate. The putative isochorismate hydrolase OaIHL from O. antarctica possesses the typical catalytic triad for IHL proteins. Its active center resembles those IHLs with a D–K–C catalytic triad, rather than those variants with a D–K–X triad. OaIHL shares some structural and sequential features with other members of the IHL superfamily. In silico docking results showed that despite small differences in active site composition, isochorismate binds to in the structure of OaIHL in a similar mode to its binding in phenazine biosynthesis protein PhzD (PDB code 1NF8).     

Crystal structure of methyl parathion hydrolase from Pseudomonas sp. WBC-3

Methyl parathion hydrolase (MPH, E.C.3.1.8.1), isolated from the soil-dwelling bacterium Pseudomonas sp. WBC-3, is a Zn(II)-containing enzyme that catalyzes the degradation of the organophosphate pesticide methyl parathion. We have determined the structure of MPH from Pseudomonas sp. WBC-3 to 2.4 angstroms resolution. The enzyme is dimeric and each subunit contains a mixed hybrid binuclear zinc center, in which one of the zinc ions is replaced by cadmium. In both subunits, the more solvent-exposed beta-metal ion is substituted for Cd2+ due to high cadmium concentration in the crystallization condition. Both ions are surrounded by ligands in an octahedral arrangement. The ions are separated by 3.5 angstroms and are coordinated by the amino acid residues His147, His149, Asp151, His152, His234 and His302 and a water molecule. Asp255 and a water molecule serve to bridge the zinc ions together. MPH is homologous with other metallo-beta-lactamases but does not show any similarity to phosphotriesterase that can also catalyze the degradation of methyl parathion with lower rate, despite the lack of sequence homology. Trp179, Phe196 and Phe119 form an aromatic cluster at the entrance of the catalytic center. Replacement of these three amino acids by alanine resulted in a significant increase of K(m) and loss of catalytic activity, indicating that the aromatic cluster has an important role to facilitate affinity of enzyme to the methyl parathion substrates.     

Crystal structure of Homo sapiens PTD012 reveals a zinc-containing hydrolase fold

The human protein PTD012 is the longer product of an alternatively spliced gene and was described to be localized in the nucleus. The X-ray structure analysis at 1.7 A resolution of PTD012 through SAD phasing reveals a monomeric protein and a novel fold. The shorter splice form was also studied and appears to be unfolded and non-functional. The structure of PTD012 displays an alphabetabetaalpha four-layer topology. A metal ion residing between the central beta-sheets is partially coordinated by three histidine residues. X-ray absorption near-edge structure (XANES) analysis identifies the PTD012-bound ion as Zn(2+). Tetrahedral coordination of the ion is completed by the carboxylate oxygen atom of an acetate molecule taken up from the crystallization buffer. The binding of Zn(2+) to PTD012 is reminiscent of zinc-containing enzymes such as carboxypeptidase, carbonic anhydrase, and beta-lactamase. Biochemical assays failed to demonstrate any of these enzyme activities in PTD012. However, PTD012 exhibits ester hydrolase activity on the substrate p-nitrophenyl acetate.     

Crystal structure and mechanism of catalysis of a pyrazinamidase from Pyrococcus horikoshii.

Bacterial pyrazinamidase (PZAase)/nicotinamidase converts pyrazinamide (PZA) to ammonia and pyrazinoic acid, which is active against Mycobacterium tuberculosis. Loss of PZAase activity is the major mechanism of pyrazinamide-resistance by M. tuberculosis. We have determined the crystal structure of the gene product of Pyrococcus horikoshii 999 (PH999), a PZAase, and its complex with zinc ion by X-ray crystallography. The overall fold of PH999 is similar to that of N-carbamoylsarcosine amidohydrolase (CSHase) of Arthrobacter sp. and YcaC of Escherichia coli, a protein with unknown physiological function. The active site of PH999 was identified by structural features that are also present in the active sites of CSHase and YcaC: a triad (D10, K94, and C133) and a cis-peptide (between V128 and A129). Surprisingly, a metal ion-binding site was revealed in the active site and subsequently confirmed by crystal structure of PH999 in complex with Zn(2+). The roles of the triad, cis-peptide, and metal ion in the catalysis are proposed. Because of extensive homology between PH999 and PZAase of M. tuberculosis (37% sequence identity), the structure of PH999 provides a structural basis for understanding PZA-resistance by M. tuberculosis harboring PZAase mutations.     

Crystal structure of beta-D-xylosidase from Thermoanaerobacterium saccharolyticum, a family 39 glycoside hydrolase

1,4-beta-D-Xylan is the major component of plant cell-wall hemicelluloses. beta-D-Xylosidases are involved in the breakdown of xylans into xylose and belong to families 3, 39, 43, 52, and 54 of glycoside hydrolases. Here, we report the first crystal structure of a member of family 39 glycoside hydrolase, i.e. beta-D-xylosidase from Thermoanaerobacterium saccharolyticum strain B6A-RI. This study also represents the first structure of any beta-xylosidase of the above five glycoside hydrolase families. Each monomer of T. saccharolyticum beta-xylosidase comprises three distinct domains; a catalytic domain of the canonical (beta/alpha)(8)-barrel fold, a beta-sandwich domain, and a small alpha-helical domain. We have determined the structure in two forms: D-xylose-bound enzyme and a covalent 2-deoxy-2-fluoro-alpha-D-xylosyl-enzyme intermediate complex, thus providing two snapshots in the reaction pathway. This study provides structural evidence for the proposed double displacement mechanism that involves a covalent intermediate. Furthermore, it reveals possible functional roles for His228 as the auxiliary acid/base and Glu323 as a key residue in substrate recognition.     

Inhibition of S-adenosylhomocysteine hydrolase by acyclic sugar adenosine analogue D-eritadenine. Crystal structure of S-adenosylhomocysteine hydrolase complexed with D-eritadenine

D-eritadenine (DEA) is a potent inhibitor (IC(50) = 7 nm) of S-adenosyl-l-homocysteine hydrolase (AdoHcyase). Unlike cyclic sugar Ado analogue inhibitors, including mechanism-based inhibitors, DEA is an acyclic sugar Ado analogue, and the C2' and C3' have opposite chirality to those of the cyclic sugar Ado inhibitors. Crystal structures of DEA alone and in complex with AdoHcyase have been determined to elucidate the DEA binding scheme to AdoHcyase. The DEA-complexed structure has been analyzed by comparing it with two structures of AdoHcyase complexed with cyclic sugar Ado analogues. The DEA-complexed structure has a closed conformation, and the DEA is located near the bound NAD(+). However, a UV absorption measurement shows that DEA is not oxidized by the bound NAD(+), indicating that the open-closed conformational change of AdoHcyase is due to the substrate/inhibitor binding, not the oxidation state of the bound NAD. The adenine ring of DEA is recognized by four essential hydrogen bonds as observed in the cyclic sugar Ado complexes. The hydrogen bond network around the acyclic sugar moiety indicates that DEA is more tightly connected to the protein than the cyclic sugar Ado analogues. The C3'-H of DEA is pointed toward C4 of the bound NAD(+) (C3'...C4 = 3.7 A), suggesting some interaction between DEA and NAD(+). By placing DEA into the active site of the open structure, the major forces to stabilize the closed conformation of AdoHcyase are identified as the hydrogen bonds between the backbone of His-352 and the adenine ring, and the C3'-H...C4 interaction. DEA has been believed to be an inactivator of AdoHcyase, but this study indicates that DEA is a reversible inhibitor. On the basis of the complexed structure, selective inhibitors of AdoHcyase have been designed.     

Dermorphin tetra- and heptapeptide analogues containing a [3,4] amide bond replacement by a carbon-carbon double and single bond.

Seven dermorphin hepta- and tetrapeptide analogues containing [3,4] amide bond replacement by a carbon-carbon double and single bond were prepared. 1H NMR studies of the pseudoheptapeptide in DMSO indicate the presence of extended conformations with stacking of the side chains in the N-terminal part and an inverse gamma-turn around Ser7 in the conformational equilibrium. The binding data show that the affinity of the analogues for the mu-receptor is only slightly diminished in the D-Ala2 series and is more affected in the D-Arg2 series. Since the Gly4NH is not present in these compounds we conclude that this NH is not required to stabilize the bioactive conformation nor is it directly involved in binding to the receptor.     

Crystal structure of Aspergillus niger isopullulanase, a member of glycoside hydrolase family 49

An isopullulanase (IPU) from Aspergillus niger ATCC9642 hydrolyzes α-1,4-glucosidic linkages of pullulan to produce isopanose. Although IPU does not hydrolyze dextran, it is classified into glycoside hydrolase family 49 (GH49), major members of which are dextran-hydrolyzing enzymes. IPU is highly glycosylated, making it difficult to obtain its crystal. We used endoglycosidase H_f to cleave the N-linked oligosaccharides of IPU, and we here determined the unliganded and isopanose-complexed forms of IPU, both solved at 1.7-Å resolution. IPU is composed of domains N and C joined by a short linker, with electron density maps for 11 or 12 N-acetylglucosamine residues per molecule. Domain N consists of 13 β-strands and forms a β-sandwich. Domain C, where the active site is located, forms a right-handed β-helix, and the lengths of the pitches of each coil of the β-helix are similar to those of GH49 dextranase and GH28 polygalacturonase. The entire structure of IPU resembles that of a GH49 enzyme, Penicillium minioluteum dextranase (Dex49A), despite a difference in substrate specificity. Compared with the active sites of IPU and Dex49A, the amino acid residues participating in subsites + 2 and + 3 are not conserved, and the glucose residues of isopanose bound to IPU completely differ in orientation from the corresponding glucose residues of isomaltose bound to Dex49A. The shape of the catalytic cleft characterized by the seventh coil of the β-helix and a loop from domain N appears to be critical in determining the specificity of IPU for pullulan.     

Divergence of chemical function in the alkaline phosphatase superfamily: structure and mechanism of the P-C bond cleaving enzyme phosphonoacetate hydrolase

Phosphonates constitute a class of natural products that mimic the properties of the more common organophosphate ester metabolite yet are not readily degraded owing to the direct linkage of the phosphorus atom to the carbon atom. Phosphonate hydrolases have evolved to allow bacteria to utilize environmental phosphonates as a source of carbon and phosphorus. The work reported in this paper examines one such enzyme, phosphonoacetate hydrolase. By using a bioinformatic approach, we circumscribed the biological range of phosphonoacetate hydrolase to a select group of bacterial species from different classes of Proteobacteria. In addition, using gene context, we identified a novel 2-aminoethylphosphonate degradation pathway in which phosphonoacetate hydrolase is a participant. The X-ray structure of phosphonoformate-bound phosphonoacetate hydrolase was determined to reveal that this enzyme is most closely related to nucleotide pyrophosphatase/diesterase, a promiscuous two-zinc ion metalloenzyme of the alkaline phosphatase enzyme superfamily. The X-ray structure and metal ion specificity tests showed that phosphonoacetate hydrolase is also a two-zinc ion metalloenzyme. By using site-directed mutagenesis and (32)P-labeling strategies, the catalytic nucleophile was shown to be Thr64. A structure-guided, site-directed mutation-based inquiry of the catalytic contributions of active site residues identified Lys126 and Lys128 as the most likely candidates for stabilization of the aci-carboxylate dianion leaving group. A catalytic mechanism is proposed which combines Lys12/Lys128 leaving group stabilization with zinc ion activation of the Thr64 nucleophile and the substrate phosphoryl group.     

Crystal structure of glycoside hydrolase family 78 alpha-L-Rhamnosidase from Bacillus sp. GL1

α-L-Rhamnosidase (EC 3.2.1.40) catalyzes the hydrolytic release of rhamnose from polysaccharides and glycosides. Bacillus sp. GL1 α-L-rhamnosidase (RhaB), a member of glycoside hydrolase (GH) family 78, is responsible for degrading the bacterial biofilm gellan, and also functions as a debittering agent for citrus fruit in the food and beverage industries through the release of rhamnose from plant glycoside, naringin. The X-ray crystal structure of RhaB was determined by single-wavelength anomalous diffraction using a selenomethionine derivative and refined at 1.9 Å resolution with a final R-factor of 18.2%. As is seen in the homodimeric form of the active enzyme, the structure of RhaB in crystal packing is a homodimer containing 1908 amino acids (residues 3-956), 43 glycerol molecules, four calcium ions, and 1755 water molecules. The overall structure consists of five domains, four of which are β-sandwich structures designated as domains N, D1, D2, and C, and an (α/α)₆-barrel structure designated as domain A. Structural comparison by DALI showed that RhaB shares its highest level of structural similarity with chitobiose phosphorylase (Z score of 25.3). The structure of RhaB in complex with the reaction product rhamnose (inhibitor constant, K_i = 1.8 mM) was also determined and refined at 2.1 Å with a final R-factor of 19.5%. Rhamnose is bound to the deep cleft of the (α/α)₆-barrel domain, as is seen in the clan-L GHs. Several negatively charged residues, such as Asp567, Glu572, Asp579, and Glu841, conserved in GH family 78 enzymes, interact with rhamnose, and RhaB mutants of these residues have drastically reduced enzyme activity, indicating that the residues are crucial for enzyme catalysis and/or substrate binding. To our knowledge, this is the first report on the determination of the crystal structure of α-L-rhamnosidase and identification of its clan-L (α/α)₆-barrel as a catalytic domain.     

Three-dimensional structure of nylon hydrolase and mechanism of nylon-6 hydrolysis

We performed x-ray crystallographic analyses of the 6-aminohexanoate oligomer hydrolase (NylC) from Agromyces sp. at 2.0 Å-resolution. This enzyme is a member of the N-terminal nucleophile hydrolase superfamily that is responsible for the degradation of the nylon-6 industry byproduct. We observed four identical heterodimers (27 kDa + 9 kDa), which resulted from the autoprocessing of the precursor protein (36 kDa) and which constitute the doughnut-shaped quaternary structure. The catalytic residue of NylC was identified as the N-terminal Thr-267 of the 9-kDa subunit. Furthermore, each heterodimer is folded into a single domain, generating a stacked αββα core structure. Amino acid mutations at subunit interfaces of the tetramer were observed to drastically alter the thermostability of the protein. In particular, four mutations (D122G/H130Y/D36A/E263Q) of wild-type NylC from Arthrobacter sp. (plasmid pOAD2-encoding enzyme), with a heat denaturation temperature of T_m = 52 °C, enhanced the protein thermostability by 36 °C (T_m = 88 °C), whereas a single mutation (G111S or L137A) decreased the stability by ∼10 °C. We examined the enzymatic hydrolysis of nylon-6 by the thermostable NylC mutant. Argon cluster secondary ion mass spectrometry analyses of the reaction products revealed that the major peak of nylon-6 (m/z 10,000–25,000) shifted to a smaller range, producing a new peak corresponding to m/z 1500–3000 after the enzyme treatment at 60 °C. In addition, smaller fragments in the soluble fraction were successively hydrolyzed to dimers and monomers. Based on these data, we propose that NylC should be designated as nylon hydrolase (or nylonase). Three potential uses of NylC for industrial and environmental applications are also discussed.     

Crystal structure of a human peptidyl-tRNA hydrolase reveals a new fold and suggests basis for a bifunctional activity

Peptidyl-tRNA hydrolase (Pth) activity releases tRNA from the premature translation termination product peptidyl-tRNA. Two different enzymes have been reported to encode such activity, Pth present in bacteria and eukaryotes and Pth2 present in archaea and eukaryotes. Here we report the crystallographic structure of the Homo sapiens Pth2 at a 2.0-A resolution as well as its catalytic properties. In contrast to the structure of Escherichia coli Pth, H. sapiens Pth2 has an alpha/beta fold with a four-stranded antiparallel beta-sheet in its core surrounded by two alpha-helices on each side. This arrangement of secondary structure elements generates a fold not previously reported. Its catalytic efficiency is comparable with that reported for the archaeal Sulfolobus solfataricus Pth2 and higher than that of the bacterial E. coli Pth. Several lines of evidence target the active site to two close loops with highly conserved residues. This active site architecture is unrelated to that of E. coli Pth. In addition, intermolecular contacts in the crystal asymmetric unit cell suggest a likely surface for protein-protein interactions related to the Pth2-mediated apoptosis.     

Crystal structure at 1.2 A resolution and active site mapping of Escherichia coli peptidyl-tRNA hydrolase.

Peptidyl-tRNA hydrolase activity from Escherichia coli ensures the recycling of peptidyl-tRNAs produced through abortion of translation. This activity, which is essential for cell viability, is carried out by a monomeric protein of 193 residues. The structure of crystalline peptidyl-tRNA hydrolase could be solved at 1.2 A resolution. It indicates a single alpha/beta globular domain built around a twisted mixed beta-sheet, similar to the central core of an aminopeptidase from Aeromonas proteolytica. This similarity allowed the characterization by site-directed mutagenesis of several residues of the active site of peptidyl-tRNA hydrolase. These residues, strictly conserved among the known peptidyl-tRNA hydrolase sequences, delineate a channel which, in the crystal, is occupied by the C-end of a neighbouring peptidyl-tRNA hydrolase molecule. Hence, several main chain atoms of three residues belonging to one peptidyl-tRNA hydrolase polypeptide establish contacts inside the active site of another peptidyl-tRNA hydrolase molecule. Such an interaction is assumed to represent the formation of a complex between the enzyme and one product of the catalysed reaction.     

Crystal structure of S-adenosyl-L-homocysteine hydrolase from the human malaria parasite Plasmodium falciparum

The human malaria parasite Plasmodium falciparum is responsible for the death of more than a million people each year. The emergence of strains of malarial parasite resistant to conventional drug therapy has stimulated searches for antimalarials with novel modes of action. S-Adenosyl-L-homocysteine hydrolase (SAHH) is a regulator of biological methylations. Inhibitors of SAHH affect the methylation status of nucleic acids, proteins, and small molecules. P.falciparum SAHH (PfSAHH) inhibitors are expected to provide a new type of chemotherapeutic agent against malaria. Despite the pressing need to develop selective PfSAHH inhibitors as therapeutic drugs, only the mammalian SAHH structures are currently available. Here, we report the crystal structure of PfSAHH complexed with the reaction product adenosine (Ado). Knowledge of the structure of the Ado complex in combination with a structural comparison with Homo sapiens SAHH (HsSAHH) revealed that a single substitution between the PfSAHH (Cys59) and HsSAHH (Thr60) accounts for the differential interactions with nucleoside inhibitors. To examine roles of the Cys59 in the interactions with nucleoside inhibitors, a mutant PfSAHH was prepared. A replacement of Cys59 by Thr results in mutant PfSAHH, which shows HsSAHH-like nucleoside inhibitor sensitivity. The present structure should provide opportunities to design potent and selective PfSAHH inhibitors.