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71.
Molecular dissection of PapD interaction with PapG reveals two chaperone-binding sites 总被引:6,自引:2,他引:4
Zheng Xu C. Hal Jones David Haslam Jerome S. Pinkner Karen Dodson Jan Kihlberg Scott J. Hultgren 《Molecular microbiology》1995,16(5):1011-1020
P pili are composite adhesive fibres that allow uropathogenic Escherichia coli to gain a foothold in the host by binding to receptors present on the uroepithalium via the adhesin PapG. The assembly of P pili requires a periplasmic chaperone, PapD, that has an immunoglobulin-like three-dimensional structure. PapD-subunit complex formation involves a conserved anchoring mechanism in the chaperone cleft and a‘molecular zippering’to the extreme C-terminus of pilus subunits. A chaperone-binding assay was developed using fusions of the C-terminus of PapG to maltose-binding protein (MBP/G fusions) to investigate whether chaperone-subunit complex formation requires additional interactions. PapD bound strongly to an MBP/G fusion containing the C-terminal 140 amino acids of PapG (MBP/G175-314) but only weakly to the MBP/G234-314 fusion containing 81 C-terminal residues, arguing that the region between residues 175-234 contains additional information that is required for strong PapD-PapG interactions. PapD was shown to interact with a PapG C-terminal truncate containing residues 1-198 but not a truncate containing residues 1-145, suggesting the presence of a second, independent PapD interactive site. Four peptides overlapping the second site region were tested for binding to PapD in vitro to further delineate this motif. Only one of the peptides synthesized was recognized by PapD. The MBP/G fusion containing both binding sites formed a tight complex with PapD in vivo and inhibited pilus assembly by preventing chaperone-subunit complex formation. 相似文献
72.
Naphthalene dioxygenase (NDO) fromPseudomonas sp strain NCIB 9816 is a multicomponent enzyme system which initiates naphthalene catabolism by catalyzing the addition of both atoms of molecular oxygen and two hydrogen atoms to the substrate to yield enantiomerically pure (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. NDO has a relaxed substrate specificity and catalyzes the dioxygenation of many related 2- and 3-ring aromatic and hydroaromatic (benzocyclic) compounds to their respectivecis-diols. Biotransformations with a diol-accumulating mutant, recombinant strains and purified enzyme components have established that in addition tocis-dihydroxylation, NDO also catalyzes a variety of other oxidations which include monohydroxylation, desaturation (dehydrogenation),O-andN-dealkylation and sulfoxidation reactions. In several cases, the absolute stereochemistry of the oxidation products formed by NDO are opposite to those formed by toluene dioxygenase (TDO). The reactions catalyzed by NDO and other microbial dioxygenases can yield specific hydroxylated compounds which can serve as chiral synthons in the preparation of a variety of compounds of interest to pharmaceutical and specialty chemical industries. We present here recent work documenting the diverse array of oxidation reactions catalyzed by NDO. The trends observed in the oxidation of a series of benzocyclic aromatic compounds are compared to those observed with TDO and provide the basis for prediction of regio- and stereospecificity in the oxidation of related substrates. Based on the types of reactions catalyzed and the biochemical characteristics of NDO, a mechanism for oxygen activation by NDO is proposed. 相似文献
73.
Gang Wu Paul G. Hitchen Maria Panico Simon J. North Mohamed-Ridha Barbouche Daniel Binet Howard R. Morris Anne Dell Stuart M. Haslam 《Glycoconjugate journal》2016,33(3):447-456
Glycans serve as important regulators of antibody activities and half-lives. IgE is the most heavily glycosylated antibody, but in comparison to other antibodies little is known about its glycan structure function relationships. We therefore describe the site specific IgE glycosylation from a patient with a novel hyper IgE syndrome linked to mutations in PGM3, which is an enzyme involved in synthesizing UDP-GlcNAc, a sugar donor widely required for glycosylation. A two-step method was developed to prepare two IgE samples from less than 1 mL of serum collected from a patient with PGM3 mutation and a patient with atopic dermatitis as a control subject. Then, a glycoproteomic strategy was used to study the site-specific glycosylation. No glycosylation was found at Asn264, whilst high mannose glycans were only detected at Asn275, tri-antennary glycans were exclusively observed at Asn99 and Asn252, and non-fucosylated complex glycans were detected at Asn99. The results showed similar glycosylation profiles between the two IgE samples. These observations, together with previous knowledge of IgE glycosylation, imply that IgE glycosylation is similarly regulated among healthy control, allergy and PGM3 related hyper IgE syndrome. 相似文献
74.
75.
Maw-Song Wang Richard D. Gandour James Rodgers John L. Haslam Richard L. Schowen 《Bioorganic chemistry》1975,4(4):392-406
The rate constants k12n for isomerization of the E1H isomer (pKa 8 in H2O) of ribonuclease-A to the E2H isomer (pKa = 6.1 in H2O), determined from proton-uptake measurements by the temperature-jump technique, in mixtures of protium and deuterium oxides (atom fraction of deuterium n), are described by the equation k12n = (733 ± 16)(1 − n + [0.46 ± 0.04]n)(1 − n + 0.69n)2sec−1 at 25°C. On the basis of the absolute magnitude of the rate constant, the magnitude of the solvent isotope effect and the proton inventory, it appears that the rate-determining step is proton transfer to a water molecule from the imidazolium form of a histidine residue, with a product-like activated complex resembling a hydronium ion. The subsequent motion of the protein structure to generate the new isomer (conformation change) must then occur in a time approaching a vibrational period. Alternative but less likely mechanisms include rate-limiting protein reorganization concerted with proton transfer to water, rate-limiting diffusion of hydronium ion away from the enzyme, or “solvation catalysis” of protein reorganization. 相似文献
76.
A method is described for isolating cyclic [(3)H]GMP from platelets preincubated with [(3)H]guanine. Collagen increased and aspirin decreased the cyclic [(3)H]GMP concentration in platelets. The results are consistent with a role for cyclic GMP in the platelet release reaction. 相似文献
77.
78.
Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme 总被引:11,自引:0,他引:11
Perryman SM; Rossana C; Deng TL; Vanin EF; Johnson LF 《Molecular biology and evolution》1986,3(4):313-321
We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a
1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45).
The open reading frame of 921 nucleotides from the first AUG to the
termination codon specifies a protein with a molecular mass of 34,962
daltons. The predicted amino acid sequence is 90% identical with that of
the human enzyme. The mouse sequence also has an extremely high degree of
similarity (as much as 55% identity) with prokaryotic thymidylate synthase
sequences, indicating that thymidylate synthase is among the most highly
conserved proteins studied to date. The similarity is especially pronounced
(as much as 80% identity) in the 44-amino-acid region encompassing the
binding site for deoxyuridylic acid. The cDNA sequence also suggests that
mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the
termination codon, UAA, is followed immediately by a poly(A) segment.
相似文献
79.
Summary The 13C values of submerged aquatic plants from contrasting but relatively defined habitats, and the 13C values of emergent, floating and submerged leaves of dimorphic aquatic plants, were measured. In many instances the 13C values of dissolved inorganic carbon in the water were also measured. Plant 13C values in the vicinity of-40 to-50 were found in rapidly flowing spring waters with carbonate 13C values of-16 to-21, consistent with the notion that species such as Fontinalis antipyretica almost exclusively assimilate free CO2 via RuP2 carboxylase. Plant 13C values in the vicinity of-10 to-15 in sluggish water with carbonate 13C values of about-5 were observed, consistent with the notion that boundary layer diffusion and/or HCO3
- uptake may determine the 13C value of submerged aquatic plants in these circumstances. Comparisons of 13C values of the same or related species growing in waters of similar carbonate 13C value but different flow rates confirmed this view; more negative 13C values were frequently associated with plants in fast moving water. In Britain, but not in Finland, the 13C values of submerged leaves of dimorphic plants were almost invariably more negative than in aerial leaves. The 13C value of carbonate from chalk streams and in acid springs indicate substantial inputs of respiratory CO2, as opposed to atmospheric carbon. The contributions of these variations in 13C of the carbon source, and of isotope fractionation in diffusion, to the 13C value of submerged parts of dimorphic plants is discussed. 相似文献
80.