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

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

RseP (YaeL), an Escherichia coli RIP protease, cleaves transmembrane sequences

Escherichia coli RseP (formerly YaeL) is believed to function as a 'regulated intramembrane proteolysis' (RIP) protease that introduces the second cleavage into anti-sigma(E) protein RseA at a position within or close to the transmembrane segment. However, neither its enzymatic activity nor the substrate cleavage position has been established. Here, we show that RseP-dependent cleavage indeed occurs within predicted transmembrane sequences of membrane proteins in vivo. Moreover, RseP catalyzed the same specificity proteolysis in an in vitro reaction system using purified components. Our in vivo and in vitro results show that RseP can cleave transmembrane sequences of some model membrane proteins that are unrelated to RseA, provided that the transmembrane region contains residues of low helical propensity. These results show that RseP has potential ability to cut a broad range of membrane protein sequences. Intriguingly, it is nevertheless recruited to the sigma(E) stress-response cascade as a specific player of RIP.     

The 1.9 A crystal structure of Escherichia coli MurG, a membrane-associated glycosyltransferase involved in peptidoglycan biosynthesis

The 1.9 A X-ray structure of a membrane-associated glycosyltransferase involved in peptidoglycan biosynthesis is reported. This enzyme, MurG, contains two alpha/beta open sheet domains separated by a deep cleft. Structural analysis suggests that the C-terminal domain contains the UDP-GlcNAc binding site while the N-terminal domain contains the acceptor binding site and likely membrane association site. Combined with sequence data from other MurG homologs, this structure provides insight into the residues that are important in substrate binding and catalysis. We have also noted that a conserved region found in many UDP-sugar transferases maps to a beta/alpha/beta/alpha supersecondary structural motif in the donor binding region of MurG, an observation that may be helpful in glycosyltransferase structure prediction. The identification of a conserved structural motif involved in donor binding in different UDP-sugar transferases also suggests that it may be possible to identify--and perhaps alter--the residues that help determine donor specificity.     

The crystal structure of the protein YhaK from Escherichia coli reveals a new subclass of redox sensitive enterobacterial bicupins

YhaK is a protein of unknown function found in low abundance in the cytosol of Escherichia coli. DNA array studies have revealed that YhaK is strongly up-regulated by nitroso-glutathione (GSNO) and also displays a 12-fold increase in expression during biofilm growth of E. coli 83972 and VR50 in human urine. We have determined the YhaK crystal structure and demonstrated that in vitro YhaK is a good marker for monitoring oxidative stresses in E. coli. The YhaK protein structure shows a bicupin fold where the two cupin domains are crosslinked with one intramolecular disulfide bond (Cys10 to Cys204). We found that the third cysteine in YhaK, Cys122, is oxidized to a sulfenic acid. Two chloride ions are found in the structure, one close to the reactive Cys122, and the other on a hydrophobic surface close to a symmetry-related molecule. There are major structural differences at the N-terminus of YhaK compared with similar structures that also display the bicupin fold (YhhW and hPirin). YhaK showed no quercetinase and peroxidase activity. However, reduced YhaK was very sensitive to reactive oxygen species (ROS). The complete, functional E. coli glutaredoxin or thioredoxin systems protected YhaK from oxidation. E. coli thioredoxin reductase and NADPH produced ROS and caused oxidation and oligomerization of reduced YhaK. Taken together, we propose that YhaK is the first of a new sub-class of bicupins that lack the canonical cupin metal-binding residues of pirins and may be involved in chloride binding and/or sensing of oxidative stress in enterobacteria.     

Crystal structure of IscA, an iron-sulfur cluster assembly protein from Escherichia coli

IscA, an 11 kDa member of the hesB family of proteins, binds iron and [2Fe-2S] clusters, and participates in the biosynthesis of iron-sulfur proteins. We report the crystal structure of the apo-protein form of IscA from Escherichia coli to a resolution of 2.3A. The crystals belong to the space group P3(2)21 and have unit cell dimensions a=b=66.104 A, c=150.167 A (alpha=beta=90 degrees, gamma=120 degrees ). The structure was solved using single-wavelength anomalous dispersion (SAD) phasing of a selenomethionyl derivative, and the IscA model was refined to R=21.4% (Rfree=25.4%). IscA exists as an (alpha1alpha2)2 homotetramer with the (alpha1alpha2) dimer comprising the asymmetric unit. Cys35, implicated in Fe-S cluster assembly, is located in a central cavity formed at the tetramer interface with the gamma-sulfur atoms of residues from the alpha1 and alpha2' monomers (and alpha1'alpha2) positioned close to one another (approximately equal 7 A). C-terminal residues 99-107 are disordered, and the exact positions of Cys99 and Cys101 could not be determined. However, computer modeling of C-terminal residues in the tetramer suggests that Cys99 and Cys101 in the alpha1 monomer and those of the alpha1' monomer (or alpha2 and alpha2') are positioned sufficiently close to coordinate [2Fe-2S] clusters between the two dimers, whereas this is not possible within the (alpha1alpha2) or (alpha1'alpha2') dimer. This symmetrical arrangement allows for binding of two [2Fe-2S] clusters on opposite sides of the tetramer. Modeling further reveals that Cys101 is positioned sufficiently close to Cys35 to allow Cys35 to participate in cluster assembly, formation, or transfer.     

E. coli chaperones DnaK,Hsp33 and Spy inhibit bacterial functional amyloid assembly

Amyloid formation is an ordered aggregation process, where β-sheet rich polymers are assembled from unstructured or partially folded monomers. We examined how two Escherichia coli cytosolic chaperones, DnaK and Hsp33, and a more recently characterized periplasmic chaperone, Spy, modulate the aggregation of a functional amyloid protein, CsgA. We found that DnaK, the Hsp70 homolog in E. coli, and Hsp33, a redox-regulated holdase, potently inhibited CsgA amyloidogenesis. The Hsp33 anti-amyloidogenesis activity was oxidation dependent, as oxidized Hsp33 was significantly more efficient than reduced Hsp33 at preventing CsgA aggregation. When soluble CsgA was seeded with preformed amyloid fibers, neither Hsp33 nor DnaK were able to efficiently prevent soluble CsgA from adopting the amyloid conformation. Moreover, both DnaK and Hsp33 increased the time that CsgA was reactive with the amyloid oligomer conformation-specific A11 antibody. Since CsgA must also pass through the periplasm during secretion, we assessed the ability of the periplasmic chaperone Spy to inhibit CsgA polymerization. Like DnaK and Hsp33, Spy also inhibited CsgA polymerization in vitro. Overexpression of Spy resulted in increased chaperone activity in periplasmic extracts and in reduced curli biogenesis in vivo. We propose that DnaK, Hsp33 and Spy exert their effects during the nucleation stages of CsgA fibrillation. Thus, both housekeeping and stress induced cytosolic and periplasmic chaperones may be involved in discouraging premature CsgA interactions during curli biogenesis.     

漆酶结构的研究进展

漆酶是一种含铜的多酚氧化酶,具有特异的氧化还原电位,能够催化氧化酚类和芳胺类化合物,同时伴随4个电子的传递,最终将O2还原成水。本文就近年来漆酶的结构及其特性的研究进展做扼要综述。     

Mechanistic implications for Escherichia coli cofactor-dependent phosphoglycerate mutase based on the high-resolution crystal structure of a vanadate complex

The structure of Escherichia coli cofactor-dependent phosphoglycerate mutase (dPGM), complexed with the potent inhibitor vanadate, has been determined to a resolution of 1.30 A (R-factor 0.159; R-free 0.213). The inhibitor is present in the active site, principally as divanadate, but with evidence of additional vanadate moieties at either end, and representing a different binding mode to that observed in the structural homologue prostatic acid phosphatase. The analysis reveals the enzyme-ligand interactions involved in inhibition of the mutase activity by vanadate and identifies a water molecule, observed in the native E.coli dPGM structure which, once activated by vanadate, may dephosphorylate the active protein. Rather than reflecting the active conformation previously observed for E.coli dPGM, the inhibited protein's conformation resembles that of the inactive dephosphorylated Saccharomyces cerevisiae dPGM. The provision of a high-resolution structure of both active and inactive forms of dPGM from a single organism, in conjunction with computational modelling of substrate molecules in the active site provides insight into the binding of substrates and the specific interactions necessary for three different activities, mutase, synthase and phosphatase, within a single active site. The sequence similarity of E.coli and human dPGMs allows us to correlate structure with clinical pathology.     

The structure of the Escherichia coli nucleoside diphosphate kinase reveals a new quaternary architecture for this enzyme family

Nucleoside diphosphate kinase (NDPK) catalyzes the transfer of gamma-phosphate from nucleoside triphosphates to nucleoside diphosphates. The subunit folding and the dimeric basic structural unit are remarkably the same for available structures but, depending on species, dimers self-associate to form hexamers or tetramers. The crystal structure of the Escherichia coli NDPK reveals a new tetrameric quaternary structure for this protein family. The two tetramers differ by the relative orientation of interacting dimers, which face either the convex or the concave side of their central sheet as in either Myxococcus xanthus (type I) or E. coli (type II), respectively. In the type II tetramer, the subunits interact by a new interface harboring a zone called the Kpn loop as in hexamers, but by the opposite face of this loop. The evolutionary conservation of the interface residues indicates that this new quaternary structure seems to be the most frequent assembly mode in bacterial tetrameric NDP kinases.     

The crystal structure of the Escherichia coli maltodextrin phosphorylase-acarbose complex

Acarbose is a naturally occurring pseudo-tetrasaccharide. It has been used in conjunction with other drugs in the treatment of diabetes where it acts as an inhibitor of intestinal glucosidases. To probe the interactions of acarbose with other carbohydrate recognition enzymes, the crystal structure of E. coli maltodextrin phosphorylase (MalP) complexed with acarbose has been determined at 2.95 A resolution and refined to crystallographic R-values of R (Rfree) = 0.241 (0.293), respectively. Acarbose adopts a conformation that is close to its major minimum free energy conformation in the MalP-acarbose structure. The acarviosine moiety of acarbose occupies sub-sites +1 and +2 and the disaccharide sub-sites +3 and +4. (The site of phosphorolysis is between sub-sites -1 and +1.) This is the first identification of sub-sites +3 and +4 of MalP. Interactions of the glucosyl residues in sub-sites +2 and +4 are dominated by carbohydrate stacking interactions with tyrosine residues. These tyrosines (Tyr280 and Tyr613, respectively, in the rabbit muscle phosphorylase numbering scheme) are conserved in all species of phosphorylase. A glycerol molecule from the cryoprotectant occupies sub-site -1. The identification of four oligosaccharide sub-sites, that extend from the interior of the phosphorylase close to the catalytic site to the exterior surface of MalP, provides a structural rationalization of the substrate selectivity of MalP for a pentasaccharide substrate. Crystallographic binding studies of acarbose with amylases, glucoamylases, and glycosyltranferases and NMR studies of acarbose in solution have shown that acarbose can adopt two different conformations. This flexibility allows acarbose to target a number of different enzymes. The two alternative conformations of acarbose when bound to different carbohydrate enzymes are discussed.     

The crystal structure of cholesteryl dihydrogen phosphate

Crystals of cholesteryl dihydrogen phosphate grown from 1,4-dioxane solution are monoclinic, space group C2 with $\text{a = 24.40, b = 6.27, c = 40.86}\text{A}\text{?}\text{and}\text{β = 102.7°}$. The asymmetric unit contains two molecules of cholesteryl phosphate CP and one dioxane molecule of the solvent. The CP molecules pack tail to tail in a bilayer structure. Within the layer they are arranged in double rows with their phosphate groups linked to ribbons by hydrogen bonds. Laterally the double strands of phosphate groups are separated by rows of dioxane molecules. The dioxane serves as hydrogen bond acceptor and as a spacer molecule that compensates the differences in cross-sectional area of the cholesteryl residue (38.4 Ų and the phosphate group (24 Ų). In the cholesterol matrix the CP molecules joined to double rows have packing contact with the smooth side of their skeleta and interdigitate with their annular methyl groups with those of molecules of the adjacent double rows. The branched cholesteryl side chains facing the bilayer center are loosely packed and show considerable disorder and/or thermal motion.     

The crystal structure of methylglyoxal synthase from Escherichia coli.

BACKGROUND: The reaction mechanism of methylglyoxal synthase (MGS) is believed to be similar to that of triosephosphate isomerase (TIM). Both enzymes utilise dihydroxyacetone phosphate (DHAP) to form an enediol(ate) phosphate intermediate as the first step of their reaction pathways. However, the second catalytic step in the MGS reaction pathway is characterized by the elimination of phosphate and collapse of the enediol(ate) to form methylglyoxal instead of reprotonation to form the isomer glyceraldehyde 3-phosphate. RESULTS: The crystal structure of MGS bound to formate and substoichiometric amounts of phosphate in the space group P6522 has been determined at 1.9 A resolution. This structure shows that the enzyme is a homohexamer composed of interacting five-stranded beta/alpha proteins, rather than the hallmark alpha/beta barrel structure of TIM. The conserved residues His19, Asp71, and His98 in each of the three monomers in the asymmetric unit bind to a formate ion that is present in the crystallization conditions. Differences in the three monomers in the asymmetric unit are localized at the mouth of the active site and can be ascribed to the presence or absence of a bound phosphate ion. CONCLUSIONS: In agreement with site-directed mutagenesis and mechanistic enzymology, the structure suggests that Asp71 acts as the catalytic base. Further, Asp20 and Asp101 are involved in intersubunit salt bridges. These salt bridges may provide a pathway for transmitting allosteric information.     

Crystal structure of a conserved hypothetical protein from Escherichia coli

The crystal structure of a conserved hypothetical protein from Escherichia coli has been determined using X-ray crystallography. The protein belongs to the Cluster of Orthologous Group COG1553 (National Center for Biotechnology Information database, NLM, NIH), for which there was no structural information available until now. Structural homology search with DALI algorism indicated that this protein has a new fold with no obvious similarity to those of other proteins with known three-dimensional structures. The protein quaternary structure consists of a dimer of trimers, which makes a characteristic cylinder shape. There is a large closed cavity with approximate dimensions of 16 Å × 16 Å × 20 Å in the center of the hexameric structure. Six putative active sites are positioned along the equatorial surface of the hexamer. There are several highly conserved residues including two possible functional cysteines in the putative active site. The possible molecular function of the protein is discussed.     

Crystal structure of IscS,a cysteine desulfurase from Escherichia coli

IscS is a widely distributed cysteine desulfurase that catalyzes the pyridoxal phosphate-dependent desulfuration of L-cysteine and plays a central role in the delivery of sulfur to a variety of metabolic pathways. We report the crystal structure of Escherichia coli IscS to a resolution of 2.1A. The crystals belong to the space group P2(1)2(1)2(1) and have unit cell dimensions a=73.70A, b=101.97A, c=108.62A (alpha=beta=gamma=90 degrees ). Molecular replacement with the Thermotoga maritima NifS model was used to determine phasing, and the IscS model was refined to an R=20.6% (R(free)=23.6%) with two molecules per asymmetric unit. The structure of E.coli IscS is similar to that of T.maritima NifS with nearly identical secondary structure and an overall backbone r.m.s. difference of 1.4A. However, in contrast to NifS a peptide segment containing the catalytic cysteine residue (Cys328) is partially ordered in the IscS structure. This segment of IscS (residues 323-335) forms a surface loop directed away from the active site pocket. Cys328 is positioned greater than 17A from the pyridoxal phosphate cofactor, suggesting that a large conformational change must occur during catalysis in order for Cys328 to participate in nucleophilic attack of a pyridoxal phosphate-bound cysteine substrate. Modeling suggests that rotation of this loop may allow movement of Cys328 to within approximately 3A of the pyridoxal phosphate cofactor.     

The X-ray structure of Escherichia coli RraA (MenG), A protein inhibitor of RNA processing

The Escherichia coli protein regulator of RNase E activity A (RraA) has recently been shown to act as a trans-acting modulator of RNA turnover in bacteria; it binds to the essential endonuclease RNase E and inhibits RNA processing in vivo and in vitro. Here, we report the 2.0A X-ray structure of RraA. The structure reveals a ring-like trimer with a central cavity of approximately 12A in diameter. Based on earlier sequence analysis, RraA had been identified as a putative S-adenosylmethionine:2-demethylmenaquinone and was annotated as MenG. However, an analysis of the RraA structure shows that the protein lacks the structural motifs usually required for methylases. Comparison of the observed fold with that of other proteins (and domains) suggests that the RraA fold is an ancient platform that has been adapted for a wide range of functions. An analysis of the amino acid sequence shows that the E.coli RraA exhibits an ancient relationship to a family of aldolases.     

Crystal structure of Escherichia coli alkanesulfonate monooxygenase SsuD

The FMNH(2)-dependent alkanesulfonate monooxygenase SsuD catalyzes the conversion of alkanesulfonates to the corresponding aldehyde and sulfite. The enzyme allows Escherichia coli to use a wide range of alkanesulfonates as sulfur sources for growth when sulfate or cysteine are not available. The structure of SsuD was solved using the multiwavelength anomalous dispersion method from only four ordered selenium sites per asymmetric unit (one site per 20,800 Da). The final model includes 328 of 380 amino acid residues and was refined to an R-factor of 23.5% (R(free)=27.5%) at 2.3A resolution. The X-ray crystal structure of SsuD shows a homotetrameric state for the enzyme, each subunit being composed of a TIM-barrel fold enlarged by four insertion regions that contribute to intersubunit interactions. SsuD is structurally related to a bacterial luciferase and an archaeal coenzyme F(420)-dependent reductase in spite of a low level of sequence identity with these enzymes. The structural relationship is not limited to the beta-barrel region; it includes most but not all extension regions and shows distinct properties for the SsuD TIM-barrel. A likely substrate-binding site is postulated on the basis of the SsuD structure presented here, results from earlier biochemical studies, and structure relatedness to bacterial luciferase. SsuD is related to other FMNH(2)-dependent monooxygenases that show distant sequence relationship to luciferase. Thus, the structure reported here provides a model for enzymes belonging to this family and suggests that they might all fold as TIM-barrel proteins.     

The structure of Escherichia coli ATP-phosphoribosyltransferase: identification of substrate binding sites and mode of AMP inhibition

ATP-phosphoribosyltransferase (ATP-PRT), the first enzyme of the histidine pathway, is a complex allosterically regulated enzyme, which controls the flow of intermediates through this biosynthetic pathway. The crystal structures of Escherichia coli ATP-PRT have been solved in complex with the inhibitor AMP at 2.7A and with product PR-ATP at 2.9A (the ribosyl-triphosphate could not be resolved). On the basis of binding of AMP and PR-ATP and comparison with type I PRTs, the PRPP and parts of the ATP-binding site are identified. These structures clearly identify the AMP as binding in the 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP)-binding site, with the adenosine ring occupying the ATP-binding site. Comparison with the recently solved Mycobacterium tuberculosis ATP-PRT structures indicates that histidine is solely responsible for the large conformational changes observed between the hexameric forms of the enzyme. The role of oligomerisation in inhibition and the structural basis for the synergistic inhibition by histidine and AMP are discussed.     

Characterization and crystal structure of Escherichia coli KDPGal aldolase

2-Keto-3-deoxy-6-phosphogluconate (KDPG) and 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolases catalyze an identical reaction differing in substrate specificity in only the configuration of a single stereocenter. However, the proteins show little sequence homology at the amino acid level. Here we investigate the determinants of substrate selectivity of these enzymes. The Escherichia coli KDPGal aldolase gene, cloned into a T7 expression vector and overexpressed in E. coli, catalyzes retro-aldol cleavage of the natural substrate, KDPGal, with values of k(cat)/K(M) and k(cat) of 1.9x10(4)M(-1)s(-1) and 4s(-1), respectively. In the synthetic direction, KDPGal aldolase efficiently catalyzes an aldol addition using a limited number of aldehyde substrates, including d-glyceraldehyde-3-phosphate (natural substrate), d-glyceraldehyde, glycolaldehyde, and 2-pyridinecarboxaldehyde. A preparative scale reaction between 2-pyridinecarboxaldehyde and pyruvate catalyzed by KDPGal aldolase produced the aldol adduct of the R stereochemistry in >99.7% ee, a result complementary to that observed using the related KDPG aldolase. The native crystal structure has been solved to a resolution of 2.4A and displays the same (alpha/beta)(8) topology, as KDPG aldolase. We have also determined a 2.1A structure of a Schiff base complex between the enzyme and its substrate. This model predicts that a single amino acid change, T161 in KDPG aldolase to V154 in KDPGal aldolase, plays an important role in determining the stereochemical course of enzyme catalysis and this prediction was borne out by site-directed mutagenesis studies. However, additional changes in the enzyme sequence are required to prepare an enzyme with both high catalytic efficiency and altered stereochemistry.