The crystal structure of a bacterial class II ketol-acid reductoisomerase: domain conservation and evolution

Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) catalyzes two steps in the biosynthesis of branched-chain amino acids. Amino acid sequence comparisons across species reveal that there are two types of this enzyme: a short form (Class I) found in fungi and most bacteria, and a long form (Class II) typical of plants. Crystal structures of each have been reported previously. However, some bacteria such as Escherichia coli possess a long form, where the amino acid sequence differs appreciably from that found in plants. Here, we report the crystal structure of the E. coli enzyme at 2.6 A resolution, the first three-dimensional structure of any bacterial Class II KARI. The enzyme consists of two domains, one with mixed alpha/beta structure, which is similar to that found in other pyridine nucleotide-dependent dehydrogenases. The second domain is mainly alpha-helical and shows strong evidence of internal duplication. Comparison of the active sites between KARI of E. coli, Pseudomonas aeruginosa, and spinach shows that most residues occupy conserved positions in the active site. E. coli KARI was crystallized as a tetramer, the likely biologically active unit. This contrasts with P. aeruginosa KARI, which forms a dodecamer, and spinach KARI, a dimer. In the E. coli KARI tetramer, a novel subunit-to-subunit interacting surface is formed by a symmetrical pair of bulbous protrusions.     

Class I tyrosyl-tRNA synthetase has a class II mode of cognate tRNA recognition

Bacterial tyrosyl-tRNA synthetases (TyrRS) possess a flexibly linked C-terminal domain of approximately 80 residues, which has hitherto been disordered in crystal structures of the enzyme. We have determined the structure of Thermus thermophilus TyrRS at 2.0 A resolution in a crystal form in which the C-terminal domain is ordered, and confirm that the fold is similar to part of the C-terminal domain of ribosomal protein S4. We have also determined the structure at 2.9 A resolution of the complex of T.thermophilus TyrRS with cognate tRNA(tyr)(G Psi A). In this structure, the C-terminal domain binds between the characteristic long variable arm of the tRNA and the anti-codon stem, thus recognizing the unique shape of the tRNA. The anticodon bases have a novel conformation with A-36 stacked on G-34, and both G-34 and Psi-35 are base-specifically recognized. The tRNA binds across the two subunits of the dimeric enzyme and, remarkably, the mode of recognition of the class I TyrRS for its cognate tRNA resembles that of a class II synthetase in being from the major groove side of the acceptor stem.     

Crystal structure of NaeI-an evolutionary bridge between DNA endonuclease and topoisomerase

NAE:I is transformed from DNA endonuclease to DNA topoisomerase and recombinase by a single amino acid substitution. The crystal structure of NAE:I was solved at 2.3 A resolution and shows that NAE:I is a dimeric molecule with two domains per monomer. Each domain contains one potential DNA recognition motif corresponding to either endonuclease or topoisomerase activity. The N-terminal domain core folds like the other type II restriction endonucleases as well as lambda-exonuclease and the DNA repair enzymes MutH and Vsr, implying a common evolutionary origin and catalytic mechanism. The C-terminal domain contains a catabolite activator protein (CAP) motif present in many DNA-binding proteins, including the type IA and type II topoisomerases. Thus, the NAE:I structure implies that DNA processing enzymes evolved from a few common ancestors. NAE:I may be an evolutionary bridge between endonuclease and DNA processing enzymes.     

Solution structure of O-glycosylated C-terminal leucine zipper domain of human salivary mucin (MUC7)

Solution structures of a 23 residue glycopeptide II (KIS* RFLLYMKNLLNRIIDDMVEQ, where * denotes the glycan Gal-beta-(1-3)-alpha-GalNAc) and its deglycosylated counterpart I derived from the C-terminal leucine zipper domain of low molecular weight human salivary mucin (MUC7) were studied using CD, NMR spectroscopy and molecular modeling. The peptide I was synthesized using the Fmoc chemistry following the conventional procedure and the glycopeptide II was synthesized incorporating the O-glycosylated building block (Nalpha-Fmoc-Ser-[Ac4-beta-D-Gal-(1,3)-Ac2-alpha-D-GalN3+ ++]-OPfp) at the appropriate position in stepwise assembly of peptide chain. Solution structures of these glycosylated and nonglycosylated peptides were studied in water and in the presence of 50% of an organic cosolvent, trifluoroethanol (TFE) using circular dichroism (CD), and in 50% TFE using two-dimensional proton nuclear magnetic resonance (2D 1H NMR) spectroscopy. CD spectra in aqueous medium indicate that the apopeptide I adapts, mostly, a beta-sheet conformation whereas the glycopeptide II assumes helical structure. This transition in the secondary structure, upon glycosylation, demonstrates that the carbohydrate moiety exerts significant effect on the peptide backbone conformation. However, in 50% TFE both the peptides show pronounced helical structure. Sequential and medium range NOEs, CalphaH chemical shift perturbations, 3JNH:CalphaH couplings and deuterium exchange rates of the amide proton resonances in water containing 50% TFE indicate that the peptide I adapts alpha-helical structure from Ile2-Val21 and the glycopeptide II adapts alpha-helical structure from Ser3-Glu22. The observation of continuous stretch of helix in both the peptides as observed by both NMR and CD spectroscopy strongly suggests that the C-terminal domain of MUC7 with heptad repeats of leucines or methionine residues may be stabilized by dimeric leucine zipper motif. The results reported herein may be invaluable in understanding the aggregation (or dimerization) of MUC7 glycoprotein which would eventually have implications in determining its structure-function relationship.     

Crystal structure of FabG4 from Mycobacterium tuberculosis reveals the importance of C-terminal residues in ketoreductase activity

Rv0242c, also known as FabG4, is a beta-ketoacyl CoA reductase in Mycobacterium tuberculosis. The crystal structure of C-terminal truncated FabG4 is solved at 2.5? resolution which shows the presence of two distinct domains, domain I and II. Domain I partially resembles "flavodoxin type domain" and the domain II is a typical "ketoacyl CoA reductase (KAR) domain". The enzyme exhibits ketoacyl CoA reductase activity by reducing acetoacyl CoA to 3-hydroxyacyl CoA in presence of NADH. Conserved catalytic triad Ser347, Tyr360, and Lys364 constitute the active site residues of the KAR domain. Presence of the Tyr and the Lys residues in the triad in a particular orientation is imperative for effective catalytic mechanism. The importance of loop I and II and the role of the C-terminal residues of KAR domain are highlighted. Comparative structural analyses clearly demonstrate that loop II is stabilized by hydrophobic interaction with C-terminal residues to sustain the orientation of Tyr360. Loop I interacts with loop II via H-bonding network to restrict the active site residue Lys364 in a catalytically favorable orientation.     

Crystal structure of heparinase II from Pedobacter heparinus and its complex with a disaccharide product

Heparinase II depolymerizes heparin and heparan sulfate glycosaminoglycans, yielding unsaturated oligosaccharide products through an elimination degradation mechanism. This enzyme cleaves the oligosaccharide chain on the nonreducing end of either glucuronic or iduronic acid, sharing this characteristic with a chondroitin ABC lyase. We have determined the first structure of a heparin-degrading lyase, that of heparinase II from Pedobacter heparinus (formerly Flavobacterium heparinum), in a ligand-free state at 2.15 A resolution and in complex with a disaccharide product of heparin degradation at 2.30 A resolution. The protein is composed of three domains: an N-terminal alpha-helical domain, a central two-layered beta-sheet domain, and a C-terminal domain forming a two-layered beta-sheet. Heparinase II shows overall structural similarities to the polysaccharide lyase family 8 (PL8) enzymes chondroitin AC lyase and hyaluronate lyase. In contrast to PL8 enzymes, however, heparinase II forms stable dimers, with the two active sites formed independently within each monomer. The structure of the N-terminal domain of heparinase II is also similar to that of alginate lyases from the PL5 family. A Zn2+ ion is bound within the central domain and plays an essential structural role in the stabilization of a loop forming one wall of the substrate-binding site. The disaccharide binds in a long, deep canyon formed at the top of the N-terminal domain and by loops extending from the central domain. Based on structural comparison with the lyases from the PL5 and PL8 families having bound substrates or products, the disaccharide found in heparinase II occupies the "+1" and "+2" subsites. The structure of the enzyme-product complex, combined with data from previously characterized mutations, allows us to propose a putative chemical mechanism of heparin and heparan-sulfate degradation.     

The three-dimensional structure of a glutathione S-transferase from the mu gene class. Structural analysis of the binary complex of isoenzyme 3-3 and glutathione at 2.2-A resolution.

The crystal structure of a mu class glutathione S-transferase (EC 2.5.1.18) from rat liver (isoenzyme 3-3) in complex with the physiological substrate glutathione (GSH) has been solved at 2.2-A resolution by multiple isomorphous replacement methods. The enzyme crystallized in the monoclinic space group C2 with unit cell dimensions of a = 87.98 A, b = 69.41 A, c = 81.34 A, and beta = 106.07 degrees. Oligonucleotide-directed site-specific mutagenesis played an important role in the solution of the structure in that the cysteine mutants C86S, C114S, and C173S were used to help locate the positions of mercuric ion sites in nonisomorphous derivatives with ethylmercuric phosphate and to align the sequence with the model derived from MIR phases. A complete model for the protein was not obtained until part of the solvent structure was interpreted. The dimer in the asymmetric unit refined to a crystallographic R = 0.171 for 19,298 data and I > or = 1.5 sigma (I). The final model consists of 4150 atoms, including all non-hydrogen atoms of 434 amino acid residues, two GSH molecules, and oxygen atoms of 474 water molecules. The dimeric enzyme is globular in shape with dimensions of 53 x 62 x 56 A. Crystal contacts are primarily responsible for conformational differences between the two subunits which are related by a noncrystallographic 2-fold axis. The structure of the type 3 subunit can be divided into two domains separated by a short linker, a smaller alpha/beta domain (domain I, residues 1-82), and a larger alpha domain (domain II, residues 90-217). Domain I contains four beta-strands which form a central mixed beta-sheet and three alpha-helices which are arranged in a beta alpha beta alpha beta beta alpha motif. Domain II is composed of five alpha-helices. Domain I can be considered the glutathione binding domain, while domain II seems to be primarily responsible for xenobiotic substrate binding. The active site is located in a deep (19-A) cavity which is composed of three relatively mobile structural elements: the long loop (residues 33-42) of domain I, the alpha 4/alpha 5 helix-turn-helix segment, and the C-terminal tail. GSH is bound at the active site in an extended conformation at one end of the beta-sheet of domain I with its backbone facing the cavity and the sulfur pointing toward the subunit to which it is bound.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structure of the periplasmic domain of Pseudomonas aeruginosa TolA: evidence for an evolutionary relationship with the TonB transporter protein

The crystal structure of the C-terminal domain III of Pseudomonas aeruginosa TolA has been determined at 1.9 A resolution. The fold is similar to that of the corresponding domain of Escherichia coli TolA, despite the limited amino acid sequence identity of the two proteins (20%). A pattern was discerned that conserves the fold of domain III within the wider TolA family and, moreover, reveals a relationship between TolA domain III and the C-terminal domain of the TonB transporter proteins. We propose that the TolA and TonB C-terminal domains have a common evolutionary origin and are related by means of domain swapping, with interesting mechanistic implications. We have also determined the overall shape of the didomain, domains II + III, of P.aeruginosa TolA by solution X-ray scattering. The molecule is monomeric-its elongated, stalk shape can accommodate the crystal structure of domain III at one end, and an elongated helical bundle within the portion corresponding to domain II. Based on these data, a model for the periplasmic domains of P.aeruginosa TolA is presented that may explain the inferred allosteric properties of members of the TolA family. The mechanisms of TolA-mediated entry of bateriophages in P.aeruginosa and E.coli are likely to be similar.     

The role of the carboxyl terminal alpha-helical coiled-coil domain in osmosensing by transporter ProP of Escherichia coli

Concentrative uptake of osmoprotectants via transporter ProP contributes to the rehydration of Escherichia coli cells that encounter high osmolality media. A member of the major facilitator superfamily, ProP is activated by osmotic upshifts in whole bacteria, in cytoplasmic membrane vesicles and in proteoliposomes prepared with the purified protein. Soluble protein ProQ is also required for full osmotic activation of ProP in vivo. ProP is differentiated from structural and functional homologues by its osmotic activation and its C-terminal extension, which is predicted to form an alpha-helical coiled-coil. A synthetic polypeptide corresponding to the C-terminus of ProP (ProP-p) formed a dimeric alpha-helical coiled-coil. A derivative of transporter ProP lacking 26 C-terminal amino acids was expressed but inactive. A derivative harbouring amino acid changes K460I, Y467I and H495I (each at the core, coiled-coil 'a' position) required a larger osmotic upshift for activation than did the wild type transporter. The same changes extended, stabilized and altered the oligomeric state of the coiled-coil formed by ProP-p. Amino acid change R488I (also at the 'a' position) further increased the magnitude of the osmotic upshift required to activate ProP, reduced the activity attained and rendered ProP activation transient. Unexpectedly, replacement R488I destabilized the coiled-coil formed by ProP-p. The activity and osmotic activation of ProP were even more strongly attenuated by helix-destabilizing change I474P. These data demonstrate that the carboxyl terminal domain of ProP can form a homodimeric alpha-helical coiled-coil with unusual properties. They implicate the C-terminal domain in the osmotic activation of ProP.     

Implications of secondary structure prediction and amino acid sequence comparison of class I and class II phosphoribosyl diphosphate synthases on catalysis, regulation, and quaternary structure

Spinach 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) synthase isozyme 4 was synthesized in Escherichia coli and purified to near homogeneity. The activity of the enzyme is independent of P(i); it is inhibited by ADP in a competitive manner, indicating a lack of an allosteric site; and it accepts ATP, dATP, GTP, CTP, and UTP as diphosphoryl donors. All of these properties are characteristic for class II PRPP synthases. K(m) values for ATP and ribose 5-phosphate are 77 and 48 microM, respectively. Gel filtration reveals a molecular mass of the native enzyme of approximately 110 kD, which is consistent with a homotrimer. Secondary structure prediction shows that spinach PRPP synthase isozyme 4 has a general folding similar to that of Bacillus subtilis class I PRPP synthase, for which the three-dimensional structure has been solved, as the position and extent of helices and beta-sheets of the two enzymes are essentially conserved. Amino acid sequence comparison reveals that residues of class I PRPP synthases interacting with allosteric inhibitors are not conserved in class II PRPP synthases. Similarly, residues important for oligomerization of the B. subtilis enzyme show little conservation in the spinach enzyme. In contrast, residues of the active site of B. subtilis PRPP synthase show extensive conservation in spinach PRPP synthase isozyme 4.     

Insights into catalysis by a knotted TrmD tRNA methyltransferase

The crystal structure of Escherichia coli tRNA (guanosine-1) methyltransferase (TrmD) complexed with S-adenosyl homocysteine (AdoHcy) has been determined at 2.5A resolution. TrmD, which methylates G37 of tRNAs containing the sequence G36pG37, is a homo-dimer. Each monomer consists of a C-terminal domain connected by a flexible linker to an N-terminal AdoMet-binding domain. The two bound AdoHcy moieties are buried at the bottom of deep clefts. The dimer structure appears integral to the formation of the catalytic center of the enzyme and this arrangement strongly suggests that the anticodon loop of tRNA fits into one of these clefts for methyl transfer to occur. In addition, adjacent hydrophobic sites in the cleft delineate a defined pocket, which may accommodate the GpG sequence during catalysis. The dimer contains two deep trefoil peptide knots and a peptide loop extending from each knot embraces the AdoHcy adenine ring. Mutational analyses demonstrate that the knot is important for AdoMet binding and catalytic activity, and that the C-terminal domain is not only required for tRNA binding but plays a functional role in catalytic activity.     

The C-terminal domain of dimeric serine hydroxymethyltransferase plays a key role in stabilization of the quaternary structure and cooperative unfolding of protein: domain swapping studies with enzymes having high sequence identity

The serine hydroxymethyltransferase from Bacillus subtilis (bsSHMT) and B. stearothermophilus (bstSHMT) are both homodimers and share approximately 77% sequence identity; however, they show very different thermal stabilities and unfolding pathways. For investigating the role of N- and C-terminal domains in stability and unfolding of dimeric SHMTs, we have swapped the structural domains between bs- and bstSHMT and generated the two novel chimeric proteins bsbstc and bstbsc, respectively. The chimeras had secondary structure, tyrosine, and pyridoxal-5'-phosphate microenvironment similar to that of the wild-type proteins. The chimeras showed enzymatic activity slightly higher than that of the wild-type proteins. Interestingly, the guanidium chloride (GdmCl)-induced unfolding showed that unlike the wild-type bsSHMT, which undergoes dissociation of native dimer into monomers at low guanidium chloride (GdmCl) concentration, resulting in a non-cooperative unfolding of enzyme, its chimera bsbstc, having the C-terminal domain of bstSHMT was resistant to low GdmCl concentration and showed a GdmCl-induced cooperative unfolding from native dimer to unfolded monomer. In contrast, the wild-type dimeric bstSHMT was resistant to low GdmCl concentration and showed a GdmCl-induced cooperative unfolding, whereas its chimera bstbsc, having the C- terminal domain of bsSHMT, showed dissociation of native dimer into monomer at low GdmCl concentration and a GdmCl-induced non-cooperative unfolding. These results clearly demonstrate that the C-terminal domain of dimeric SHMT plays a vital role in stabilization of the oligomeric structure of the native enzyme hence modulating its unfolding pathway.     

The tetramerization domain of the Mnt repressor consists of two right-handed coiled coils.

The tetrameric Mnt repressor is involved in the genetic switch between the lysogenic and lytic growth of Salmonella bacteriophage P22. The solution structure of its C-terminal tetramerization domain, which holds together the two dimeric DNA-binding domains, has been determined by NMR spectroscopy. This structure reveals an assembly of four alpha-helical subunits, consisting of a dimer of two antiparallel coiled coils with a unique right-handed twist. The superhelical winding is considerably stronger and the interhelical separation closer than those found in the well-known left-handed coiled coils in fibrous proteins and leucine zippers. An unusual asymmetry arises between the two monomers that comprise one right-handed coiled coil. A difference in the packing to the adjacent monomer of the other coiled coil occurs with an offset of two helical turns. The two asymmetric monomers within each coiled coil interconvert on a time scale of seconds. Both with respect to symmetry and handedness of helical packing, the C2 symmetric four-helix bundle of Mnt differs from other oligomerization domains that assemble DNA-binding modules, such as that in the tumor suppressor p53 and the E. coli lac repressor.     

Developmentally regulated cytokeratin gene in Xenopus laevis.

We have determined the sequence of cloned cDNAs derived from a 1,665-nucleotide mRNA which transiently accumulates during Xenopus laevis embryogenesis. Computer analysis of the deduced amino acid sequence revealed that this mRNA encodes a 47-kilodalton type I intermediate filament subunit, i.e., a cytokeratin. As is common to all intermediate filament subunits so far examined, the predicted polypeptide, named XK70, contains N- and C-terminal domains flanking a central alpha-helical rod domain. The overall amino acid homology between XK70 and a human 50-kilodalton type I keratin is 47%; homology within the alpha-helical domain is 57%. The N-terminal domain, which is not completely contained in our cDNAs, is basic, contains 42% serine plus alanine, and includes five copies of a six-amino-acid repeating unit. The C-terminal domain has a high alpha-helical content and contains a region with sequence homology to the C-terminal domains of other type I and type III intermediate filament proteins. We suggest that different keratin filament subtypes may have different functional roles during amphibian oogenesis and embryogenesis.     

The cytochrome c domain of dimeric cytochrome cd(1) of Paracoccus pantotrophus can be produced at high levels as a monomeric holoprotein using an improved c-type cytochrome expression system in Escherichia coli

Cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus is a dimer; within each monomer there is a largely alpha-helical domain that contains the c-type cytochrome centre. The structure of this domain changes significantly upon reduction of the heme iron, for which the ligands change from His17/His69 to Met106/His69. Overproduction, using an improved Escherichia coli expression system, of this c-type cytochrome domain as an independent monomer is reported here. The properties of the independent domain are compared with those when it is part of dimeric holo or semi-apo cytochrome cd(1).     

Crystal structure of a prokaryotic aspartyl tRNA-synthetase.

The crystal structure of Thermus thermophilus aspartyl tRNA-synthetase (AspRS) refined at 2.5 A resolution is described. This molecular structure is a textbook illustration of the modular organization of aminoacyl-tRNA synthetases. In addition to the three domains found in yeast AspRS, each monomer exhibits a module specific to prokaryotic enzymes, which corresponds to a helix-turn-helix motif in yeast AspRS, a domain implicated in the stabilization of the complex with tRNA. Its topology matches that of the histidine-containing phosphocarrier HPr which has been linked recently to another group of proteins containing the ferredoxin fold. We propose a more extensive alignment of these folds, which involves a circular permutation of the sequences and changes the point of entry of the whole domain. The C-terminal extension, another prokaryotic characteristic, leads to a significant increase in the network of interaction at the dimer interface. Some potential communication pathways suggest how a transfer of information between the two active sites of the homodimer might occur. Most of the residues involved belong to the class II-specific motifs in correlation with the dimeric state of nearly all class II enzymes. The T. thermophilus enzyme exhibits some features not found in any of the six other known AspRSs from mesophilic organisms.     

Conserved structural elements in glutathione transferase homologues encoded in the genome of Escherichia coli

Multiple sequence alignments of the eight glutathione (GSH) transferase homologues encoded in the genome of Escherichia coli were used to define a consensus sequence for the proteins. The consensus sequence was analyzed in the context of the three-dimensional structure of the gst gene product (EGST) obtained from two different crystal forms of the enzyme. The enzyme consists of two domains. The N-terminal region (domain I) has a thioredoxin-like alpha/beta-fold, while the C-terminal domain (domain II) is all alpha-helical. The majority of the consensus residues (12/17) reside in the N-terminal domain. Fifteen of the 17 residues are involved in hydrophobic core interactions, turns, or electrostatic interactions between the two domains. The results suggest that all of the homologues retain a well-defined group of structural elements both in and between the N-terminal alpha/beta domain and the C-terminal domain. The conservation of two key residues for the recognition motif for the gamma-glutamyl-portion of GSH indicates that the homologues may interact with GSH or GSH analogues such as glutathionylspermidine or alpha-amino acids. The genome context of two of the homologues forms the basis for a hypothesis that the b2989 and yibF gene products are involved in glutathionylspermidine and selenium biochemistry, respectively.