NMR structure of YcgL, a conserved protein from Escherichia coli representing the DUF709 family, with a novel alpha/beta/alpha sandwich fold.
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Amino Acid Sequence↗
Biomedical subjects
Miroslaw Cygler
Publications and source records attributed to Miroslaw Cygler.
Quantitative continuous assay for hyaluronan synthase.
A rapid, continuous, and convenient three-enzyme coupled UV absorption assay was developed to quantitate the glucuronic acid and N-acetylglucosamine transferase activities of hyaluronan synthase from Pasteurella multocida (PmHAS). Activity was measured by coupling the UDP produced from the PmHAS-catalyzed transfer of UDP-GlcNAc and UDP-GlcUA to a hyaluronic acid tetrasaccharide primer with the oxidation of NADH. Using a fluorescently labeled primer, the products were characterized by gel electrophoresis. Our results show that a truncated soluble form of recombinant PmHAS (residues 1-703) can catalyze the glycosyl transfers in a time- and concentration-dependent manner. The assay can be used to determine kinetic parameters, inhibition constants, and mechanistic aspects of this enzyme. In addition, it can be used to quantify PmHAS during purification of the enzyme from culture media.
Fluorescent Dyes↗
The architecture of the multisubunit TRAPP I complex suggests a model for vesicle tethering.
Transport protein particle (TRAPP) I is a multisubunit vesicle tethering factor composed of seven subunits involved in ER-to-Golgi trafficking. The functional mechanism of the complex and how the subunits interact to form a functional unit are unknown. Here, we have used a multidisciplinary approach that includes X-ray crystallography, electron microscopy, biochemistry, and yeast genetics to elucidate the architecture of TRAPP I. The complex is organized through lateral juxtaposition of the subunits into a flat and elongated particle. We have also localized the site of guanine nucleotide exchange activity to a highly conserved surface encompassing several subunits. We propose that TRAPP I attaches to Golgi membranes with its large flat surface containing many highly conserved residues and forms a platform for protein-protein interactions. This study provides the most comprehensive view of a multisubunit vesicle tethering complex to date, based on which a model for the function of this complex, involving Rab1-GTP and long, coiled-coil tethers, is presented.
Amino Acids↗
Structural snapshots of Escherichia coli histidinol phosphate phosphatase along the reaction pathway.
HisB from Escherichia coli is a bifunctional enzyme catalyzing the sixth and eighth steps of l-histidine biosynthesis. The N-terminal domain (HisB-N) possesses histidinol phosphate phosphatase activity, and its crystal structure shows a single domain with fold similarity to the haloacid dehalogenase (HAD) enzyme family. HisB-N forms dimers in the crystal and in solution. The structure shows the presence of a structural Zn(2+) ion stabilizing the conformation of an extended loop. Two metal binding sites were also identified in the active site. Their presence was further confirmed by isothermal titration calorimetry. HisB-N is active in the presence of Mg(2+), Mn(2+), Co(2+), or Zn(2+), but Ca(2+) has an inhibitory effect. We have determined structures of several intermediate states corresponding to snapshots along the reaction pathway, including that of the phosphoaspartate intermediate. A catalytic mechanism, different from that described for other HAD enzymes, is proposed requiring the presence of the second metal ion not found in the active sites of previously characterized HAD enzymes, to complete the second half-reaction. The proposed mechanism is reminiscent of two-Mg(2+) ion catalysis utilized by DNA and RNA polymerases and many nucleases. The structure also provides an explanation for the inhibitory effect of Ca(2+).
Amino Acid Sequence↗
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.
Acid Anhydride Hydrolases↗
Domain organization and crystal structure of the catalytic domain of E.coli RluF, a pseudouridine synthase that acts on 23S rRNA.
Pseudouridine synthases catalyze the isomerization of uridine to pseudouridine (Psi) in rRNA and tRNA. The pseudouridine synthase RluF from Escherichia coli (E.C. 4.2.1.70) modifies U2604 in 23S rRNA, and belongs to a large family of pseudouridine synthases present in all kingdoms of life. Here we report the domain architecture and crystal structure of the catalytic domain of E.coli RluF at 2.6A resolution. Limited proteolysis, mass spectrometry and N-terminal sequencing indicate that RluF has a distinct domain architecture, with the catalytic domain flanked at the N and C termini by additional domains connected to it by flexible linkers. The structure of the catalytic domain of RluF is similar to those of RsuA and TruB. RluF is a member of the RsuA sequence family of Psi-synthases, along with RluB and RluE. Structural comparison of RluF with its closest structural homologues, RsuA and TruB, suggests possible functional roles for the N-terminal and C-terminal domains of RluF.
Amino Acid Sequence↗
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.
Bacteroidetes↗
Structural and functional characterization of PseC, an aminotransferase involved in the biosynthesis of pseudaminic acid, an essential flagellar modification in Helicobacter pylori.
Helicobacter pylori flagellin is heavily glycosylated with the novel sialic acid-like nonulosonate, pseudaminic acid (Pse). The glycosylation process is essential for assembly of functional flagellar filaments and consequent bacterial motility. Because motility is a key virulence factor for this and other important pathogens, the Pse biosynthetic pathway offers potential for novel therapeutic targets. From recent NMR analyses, we determined that the conversion of UDP-alpha-D-Glc-NAc to the central intermediate in the pathway, UDP-4-amino-4,6-dideoxy-beta-L-AltNAc, proceeds by formation of UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-4-hexulose by the dehydratase/epimerase PseB (HP0840) followed with amino transfer by the aminotransferase, PseC (HP0366). The central role of PseC in the H. pylori Pse biosynthetic pathway prompted us to determine crystal structures of the native protein, its complexes with pyridoxal phosphate alone and in combination with the UDP-4-amino-4,6-dideoxy-beta-L-AltNAc product, the latter being converted to the external aldimine form in the active site of the enzyme. In the binding site, the AltNAc sugar ring adopts a 4C1 chair conformation, which is different from the predominant 1C4 form found in solution. The enzyme forms a homodimer where each monomer contributes to the active site, and these structures have permitted the identification of key residues involved in stabilization, and possibly catalysis, of the beta-L-arabino intermediate during the amino transfer reaction. The essential role of Lys183 in the catalytic event was confirmed by site-directed mutagenesis. This work presents for the first time a nucleotide-sugar aminotransferase co-crystallized with its natural ligand, and, in conjunction with the recent functional characterization of this enzyme, these results will assist in elucidating the aminotransferase reaction mechanism within the Pse biosynthetic pathway.
Amino Acid Sequence↗
An atypical approach identifies TYR234 as the key base catalyst in chondroitin AC lyase.
Chondroitin AC lyase from Flavobacterium heparinum catalyses the degradation of chondroitin by an anionic E1cb elimination mechanism that involves proton abstraction from C5 of glucuronic acid. The lyase also carries out efficient proton transfer to a sugar nitronate anion, which was designed originally as an inhibitor of the enzyme, with a second-order rate constant of kcat/Km=2.7x10(6) M(-1) s(-); this is very similar to that of the natural chondroitin substrate (kcat/Km=1.3x10(6) M(-1) s(-1)). Studies with this nitronate should therefore provide insight into the proton-transfer step (general base catalysis) within this mechanism. Indeed, the Tyr234Phe mutant of the enzyme was essentially inactive with the natural substrate and correspondingly did not catalyse proton transfer to the nitronate, thereby implicating this residue as the general base catalyst. Parallel studies designed to identify the acid catalyst were carried out by using a substrate with a 2,4-dinitrophenol leaving group that needs no acid assistance for departure. These results are consistent with Tyr234 also playing the role of acid catalyst. Not only do these studies confirm the suspected role of Tyr234, but also they validate a new methodology for identification of acid/base catalysts in lyases and epimerases of this type. In addition a structural and mechanistic rationale is provided for different active-site acid/base configurations in syn and anti lyases.
Binding Sites↗
Crystal structure of the bb' domains of the protein disulfide isomerase ERp57.
The synthesis of proteins in the endoplasmic reticulum (ER) is limited by the rate of correct disulfide bond formation. This process is carried out by protein disulfide isomerases, a family of ER proteins which includes general enzymes such as PDI that recognize unfolded proteins and others that are selective for specific proteins or classes. Using small-angle X-ray scattering and X-ray crystallography, we report the structure of a selective isomerase, ERp57, and its interactions with the lectin chaperone calnexin. Using isothermal titration calorimetry and NMR spectroscopy, we show that the b' domain of ERp57 binds calnexin with micromolar affinity through a conserved patch of basic residues. Disruption of this binding site by mutagenesis abrogates folding of RNase B in an in vitro assay. The relative positions of the ERp57 catalytic sites and calnexin binding site suggest that activation by calnexin is due to substrate recruitment rather than a direct stimulation of ERp57 oxidoreductase activity.
Amino Acid Sequence↗
The crystal structure of CREG, a secreted glycoprotein involved in cellular growth and differentiation.
The cellular repressor of E1A-stimulated genes (CREG) is a secreted glycoprotein that inhibits proliferation and enhances differentiation of human embryonal carcinoma cells. CREG binds to the cation-independent mannose 6-phosphate (M6P)/insulin-like growth factor II (IGF2) receptor (IGF2R) (M6P/IGF2R), and this receptor has been shown to be required for CREG-induced growth suppression. To better understand CREG function in cellular growth and differentiation, we solved the 3D crystal structure of this protein to 1.9-A resolution. CREG forms a tight homodimeric complex, and CREG monomers display a beta-barrel fold. The three potential glycosylation sites on CREG map to a confined patch opposite the dimer interface. Thus, dimerization of glycosylated CREG likely presents a bivalent ligand for the M6P/IGF2R. Closely related structural homologs of CREG are FMN-binding split-barrel fold proteins that bind flavin mononucleotide. Our structure shows that the putative flavin mononucleotide-binding pocket in CREG is sterically blocked by a loop and several key bulky residues. A mutant of CREG lacking a part of this loop maintained overall structure and dimerization, as well as M6P/IGF2R binding, but lost the growth suppression activity of WT CREG. Thus, analysis of a structure-based mutant of CREG revealed that binding to M6P/IGF2R, while necessary, is not sufficient for CREG-induced growth suppression. These findings indicate that CREG utilizes a known fold for a previously undescribed function [corrected]
Amino Acid Sequence↗
Identification of an Escherichia coli O157:H7 heme oxygenase with tandem functional repeats.
Heme oxygenases (HOs) catalyze the oxidation of heme to biliverdin, carbon monoxide (CO), and free iron. Iron acquisition is critical for invading microorganisms to enable survival and growth. Here we report the crystal structure of ChuS, which displays a previously uncharacterized fold and is unique compared with other characterized HOs. Despite only 19% sequence identity between the N- and C-terminal halves, these segments of ChuS represent a structural duplication, with a root-mean-square deviation of 2.1 A between the two repeats. ChuS is capable of using ascorbic acid or cytochrome P450 reductase-NADPH as electron sources for heme oxygenation. CO detection confirmed that ChuS is a HO, and we have identified it in pathogenic Escherichia coli O157:H7. Based on sequence analysis, this HO is present in many bacteria, although not in the E. coli K-12 strain. The N- and C-terminal halves of ChuS are each a functional HO.
Carbon Monoxide↗
Crystal structure of ureidoglycolate hydrolase (AllA) from Escherichia coli O157:H7.
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Amidine-Lyases↗
Crystallographic trapping of the glutamyl-CoA thioester intermediate of family I CoA transferases.
Coenzyme A transferases are involved in a broad range of biochemical processes in both prokaryotes and eukaryotes, and exhibit a diverse range of substrate specificities. The YdiF protein from Escherichia coli O157:H7 is an acyl-CoA transferase of unknown physiological function, and belongs to a large sequence family of CoA transferases, present in bacteria to humans, which utilize oxoacids as acceptors. In vitro measurements showed that YdiF displays enzymatic activity with short-chain acyl-CoAs. The crystal structures of YdiF and its complex with CoA, the first co-crystal structure for any Family I CoA transferase, have been determined and refined at 1.9 and 2.0 A resolution, respectively. YdiF is organized into tetramers, with each monomer having an open alpha/beta structure characteristic of Family I CoA transferases. Co-crystallization of YdiF with a variety of CoA thioesters in the absence of acceptor carboxylic acid resulted in trapping a covalent gamma-glutamyl-CoA thioester intermediate. The CoA binds within a well defined pocket at the N- and C-terminal domain interface, but makes contact only with the C-terminal domain. The structure of the YdiF complex provides a basis for understanding the different catalytic steps in the reaction of Family I CoA transferases.
Binding Sites↗
Crystal structure of Methanobacterium thermoautotrophicum phosphoribosyl-AMP cyclohydrolase HisI.
The metabolic pathway for histidine biosynthesis is interesting from an evolutionary perspective because of the diversity of gene organizations and protein structures involved. Hydrolysis of phosphoribosyl-AMP, the third step in the histidine biosynthetic pathway, is carried out by PR-AMP cyclohydrolase, the product of the hisI gene. The three-dimensional structure of PR-AMP cyclohydrolase from Methanobacterium thermoautotrophicum was solved and refined to 1.7 A resolution. The enzyme is a homodimer. The position of the Zn(2+)-binding site that is essential for catalysis was inferred from the positions of bound Cd(2+) ions, which were part of the crystallization medium. These metal binding sites include three cysteine ligands, two from one monomer and the third from the second monomer. The enzyme remains active when Cd(2+) is substituted for Zn(2+). The likely binding site for Mg(2+), also necessary for activity in a homologous cyclohydrolase, was also inferred from Cd(2+) positions and is comprised of aspartic acid side chains. The putative substrate-binding cleft is formed at the interface between the two monomers of the dimer. This fact, combined with the localization of the Zn(2+)-binding site, indicates that the enzyme is an obligate dimer.
Amino Acid Sequence↗
Crystal structure of Escherichia coli crotonobetainyl-CoA: carnitine CoA-transferase (CaiB) and its complexes with CoA and carnitinyl-CoA.
L-Carnitine (R-[-]-3-hydroxy-4-trimethylaminobutyrate) is found in both eukaryotic and prokaryotic cells and participates in diverse processes including long-chain fatty-acid transport and osmoprotection. The enzyme crotonobetainyl/gamma-butyrobetainyl-CoA:carnitine CoA-transferase (CaiB; E.C. 2.8.3.-) catalyzes the first step in carnitine metabolism, leading to the final product gamma-butyrobetaine. The crystal structures of Escherichia coli apo-CaiB, as well as its Asp169Ala mutant bound to CoA and to carnitinyl-CoA, have been determined and refined to 1.6, 2.4, and 2.4 A resolution, respectively. CaiB is composed of two identical circular chains that together form an intertwined dimer. Each monomer consists of a large domain, containing a Rossmann fold, and a small domain. The monomer and dimer resemble those of formyl-CoA transferase from Oxalobacter formigenes, as well as E. coli YfdW, a putative type-III CoA transferase of unknown function. The CoA cofactor-binding site is formed at the interface of the large domain of one monomer and the small domain from the second monomer. Most of the protein-CoA interactions are formed with the Rossmann fold domain. While the location of cofactor binding is similar in the three proteins, the specific CoA-protein interactions vary somewhat between CaiB, formyl-CoA transferase, and YfdW. CoA binding results in a change in the relative positions of the large and small domains compared with apo-CaiB. The observed carnitinyl-CoA product in crystals of the CaiB Asp169Ala mutant cocrystallized with crotonoyl-CoA and carnitine could result from (i) a catalytic mechanism involving a ternary enzyme-substrate complex, independent of a covalent anhydride intermediate with Asp169, (ii) a spontaneous reaction of the substrates in solution, followed by binding to the enzyme, or (iii) an involvement of another residue substituting functionally for Asp169, such as Glu23.
Apoenzymes↗
Structure of the pseudouridine synthase RsuA from Haemophilus influenzae.
The structure of the pseudouridine synthase RsuA from Haemophilus influenza, which catalyzes the conversion of uridine to pseudouridine at a single position within 16S ribosomal RNA, has been determined at 1.59 A resolution and compared with that of Escherichia coli RsuA. The H. influenza enzyme contains an N-terminal S4-like alpha3beta4 domain followed by a catalytic domain, as observed in the structure of E. coli RsuA. Whereas the individual domains of E. coli and H. influenza RsuA are structurally similar, their relative spatial disposition differs greatly between the two structures. The former displays an extended open conformation with no direct contacts between the domains, while the latter is in a closed conformation with a large interface between the two domains. Domain closure presents several basic and polar residues into a putative RNA-binding cleft. It is proposed that this relative repositioning of the S4 and catalytic domains is used to modulate the shape and size of the rRNA-binding site in RsuA and in other pseudouridine synthases possessing S4 domains.
Binding Sites↗
Crystal structure of N-succinylarginine dihydrolase AstB, bound to substrate and product, an enzyme from the arginine catabolic pathway of Escherichia coli.
The ammonia-producing arginine succinyltransferase pathway is the major pathway in Escherichia coli and related bacteria for arginine catabolism as a sole nitrogen source. This pathway consists of five steps, each catalyzed by a distinct enzyme. Here we report the crystal structure of N-succinylarginine dihydrolase AstB, the second enzyme of the arginine succinyltransferase pathway, providing the first structural insight into enzymes from this pathway. The enzyme exhibits a pseudo 5-fold symmetric alpha/beta propeller fold of circularly arranged betabetaalphabeta modules enclosing the active site. The crystal structure indicates clearly that this enzyme belongs to the amidinotransferase (AT) superfamily and that the active site contains a Cys-His-Glu triad characteristic of the AT superfamily. Structures of the complexes of AstB with the reaction product and a C365S mutant with bound the N-succinylarginine substrate suggest a catalytic mechanism that consists of two cycles of hydrolysis and ammonia release, with each cycle utilizing a mechanism similar to that proposed for arginine deiminases. Like other members of the AT superfamily of enzymes, AstB possesses a flexible loop that is disordered in the absence of substrate and assumes an ordered conformation upon substrate binding, shielding the ligand from the bulk solvent, thereby controlling substrate access and product release.
Arginine↗