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1.
The sialic acid (Sia) N-acetylneuraminic acid (Neu5Ac) and its hydroxylated derivative N-glycolylneuraminic acid (Neu5Gc) differ by one oxygen atom. CMP-Neu5Gc is synthesized from CMP-Neu5Ac, with Neu5Gc representing a highly variable fraction of total Sias in various tissues and among different species. The exception may be the brain, where Neu5Ac is abundant and Neu5Gc is reported to be rare. Here, we confirm this unusual pattern and its evolutionary conservation in additional samples from various species, concluding that brain Neu5Gc expression has been maintained at extremely low levels over hundreds of millions of years of vertebrate evolution. Most explanations for this pattern do not require maintaining neural Neu5Gc at such low levels. We hypothesized that resistance of α2-8-linked Neu5Gc to vertebrate sialidases is the detrimental effect requiring the relative absence of Neu5Gc from brain. This linkage is prominent in polysialic acid (polySia), a molecule with critical roles in vertebrate neural development. We show that Neu5Gc is incorporated into neural polySia and does not cause in vitro toxicity. Synthetic polymers of Neu5Ac and Neu5Gc showed that mammalian and bacterial sialidases are much less able to hydrolyze α2-8-linked Neu5Gc at the nonreducing terminus. Notably, this difference was not seen with acid-catalyzed hydrolysis of polySias. Molecular dynamics modeling indicates that differences in the three-dimensional conformation of terminal saccharides may partly explain reduced enzymatic activity. In keeping with this, polymers of N-propionylneuraminic acid are sensitive to sialidases. Resistance of Neu5Gc-containing polySia to sialidases provides a potential explanation for the rarity of Neu5Gc in the vertebrate brain.  相似文献   

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Plant fatty acids can be completely degraded within the peroxisomes. Fatty acid degradation plays a role in several plant processes including plant hormone synthesis and seed germination. Two multifunctional peroxisomal isozymes, MFP2 and AIM1, both with 2-trans-enoyl-CoA hydratase and l-3-hydroxyacyl-CoA dehydrogenase activities, function in mouse ear cress (Arabidopsis thaliana) peroxisomal β-oxidation, where fatty acids are degraded by the sequential removal of two carbon units. A deficiency in either of the two isozymes gives rise to a different phenotype; the biochemical and molecular background for these differences is not known. Structure determination of Arabidopsis MFP2 revealed that plant peroxisomal MFPs can be grouped into two families, as defined by a specific pattern of amino acid residues in the flexible loop of the acyl-binding pocket of the 2-trans-enoyl-CoA hydratase domain. This could explain the differences in substrate preferences and specific biological functions of the two isozymes. The in vitro substrate preference profiles illustrate that the Arabidopsis AIM1 hydratase has a preference for short chain acyl-CoAs compared with the Arabidopsis MFP2 hydratase. Remarkably, neither of the two was able to catabolize enoyl-CoA substrates longer than 14 carbon atoms efficiently, suggesting the existence of an uncharacterized long chain enoyl-CoA hydratase in Arabidopsis peroxisomes.  相似文献   

3.
Xu  Quanle  Liu  Fengjuan  Qu  Ruihong  Gillman  Jason D.  Bi  Chunxiao  Hu  Xin  Chen  Peng  Krishnan  Hari B. 《Plant Molecular Biology Reporter》2018,36(5-6):832-843
Plant Molecular Biology Reporter - Grass pea (Lathyrus sativus L.) is a unique potential crop for marginal arid regions with untapped, exceptional biotic/abiotic stress tolerance, and high protein...  相似文献   

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Pyridoxal 5′-phosphate (PLP)-dependent basic amino acid decarboxylases from the β/α-barrel-fold class (group IV) exist in most organisms and catalyze the decarboxylation of diverse substrates, essential for polyamine and lysine biosynthesis. Herein we describe the first x-ray structure determination of bacterial biosynthetic arginine decarboxylase (ADC) and carboxynorspermidine decarboxylase (CANSDC) to 2.3- and 2.0-Å resolution, solved as product complexes with agmatine and norspermidine. Despite low overall sequence identity, the monomeric and dimeric structures are similar to other enzymes in the family, with the active sites formed between the β/α-barrel domain of one subunit and the β-barrel of the other. ADC contains both a unique interdomain insertion (4-helical bundle) and a C-terminal extension (3-helical bundle) and it packs as a tetramer in the asymmetric unit with the insertions forming part of the dimer and tetramer interfaces. Analytical ultracentrifugation studies confirmed that the ADC solution structure is a tetramer. Specificity for different basic amino acids appears to arise primarily from changes in the position of, and amino acid replacements in, a helix in the β-barrel domain we refer to as the “specificity helix.” Additionally, in CANSDC a key acidic residue that interacts with the distal amino group of other substrates is replaced by Leu314, which interacts with the aliphatic portion of norspermidine. Neither product, agmatine in ADC nor norspermidine in CANSDC, form a Schiff base to pyridoxal 5′-phosphate, suggesting that the product complexes may promote product release by slowing the back reaction. These studies provide insight into the structural basis for the evolution of novel function within a common structural-fold.  相似文献   

6.
The open reading frame Rv1326c of Mycobacterium tuberculosis (Mtb) H37Rv encodes for an α-1,4-glucan branching enzyme (MtbGlgB, EC 2.4.1.18, Uniprot entry Q10625). This enzyme belongs to glycoside hydrolase (GH) family 13 and catalyzes the branching of a linear glucose chain during glycogenesis by cleaving a 1→4 bond and making a new 1→6 bond. Here, we show the crystal structure of full-length MtbGlgB (MtbGlgBWT) at 2.33-Å resolution. MtbGlgBWT contains four domains: N1 β-sandwich, N2 β-sandwich, a central (β/α)8 domain that houses the catalytic site, and a C-terminal β-sandwich. We have assayed the amylase activity with amylose and starch as substrates and the glycogen branching activity using amylose as a substrate for MtbGlgBWT and the N1 domain-deleted (the first 108 residues deleted) MtbΔ108GlgB protein. The N1 β-sandwich, which is formed by the first 105 amino acids and superimposes well with the N2 β-sandwich, is shown to have an influence in substrate binding in the amylase assay. Also, we have checked and shown that several GH13 family inhibitors are ineffective against MtbGlgBWT and MtbΔ108GlgB. We propose a two-step reaction mechanism, for the amylase activity (1→4 bond breakage) and isomerization (1→6 bond formation), which occurs in the same catalytic pocket. The structural and functional properties of MtbGlgB and MtbΔ108GlgB are compared with those of the N-terminal 112-amino acid-deleted Escherichia coli GlgB (ECΔ112GlgB).  相似文献   

7.
1Georissamenglunensissp.nov.(Fig.l)Holotype.Hight2.omm,breadth1.4mm;lengthofoperculumo.85mm,breadthofoperculumo.7Omm.Menglum,MenglaCounty(2l'O9'N,lOl'O2'E),YunnanProvince,China,July,27,l993.Paratypes:47specimens,hight2.2O-l.7Omm,breadthl.2O-1.55mm;lengthofapertureO.65-o.85mm,breadthofapertureO.7o-O.8Omm;Menglaun,MenglaCounty,YunnanProvince,China.July27,l993;May23,1994.TypespecimensaredepositedintheInstituteofZoologytheChineseAcademyofSci-ences,Beijing.1Shellsmall,thin.Whols47,we…  相似文献   

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Despite its essential role in the yeast cell wall, the exact composition of the β-(1,6)-glucan component is not well characterized. While solubilizing the cell wall alkali-insoluble fraction from a wild type strain of Saccharomyces cerevisiae using a recombinant β-(1,3)-glucanase followed by chromatographic characterization of the digest on an anion exchange column, we observed a soluble polymer that eluted at the end of the solvent gradient run. Further characterization indicated this soluble polymer to have a molecular mass of ∼38 kDa and could be hydrolyzed only by β-(1,6)-glucanase. Gas chromatographymass spectrometry and NMR (1H and 13C) analyses confirmed it to be a β-(1,6)-glucan polymer with, on average, branching at every fifth residue with one or two β-(1,3)-linked glucose units in the side chain. This polymer peak was significantly reduced in the corresponding digests from mutants of the kre genes (kre9 and kre5) that are known to play a crucial role in the β-(1,6)-glucan biosynthesis. In the current study, we have developed a biochemical assay wherein incubation of UDP-[14C]glucose with permeabilized S. cerevisiae yeasts resulted in the synthesis of a polymer chemically identical to the branched β-(1,6)-glucan isolated from the cell wall. Using this assay, parameters essential for β-(1,6)-glucan synthetic activity were defined.The cell wall of Saccharomyces cerevisiae and other yeasts contains two types of β-glucans. In the former yeast, branched β-(1,3)-glucan accounts for ∼50–55%, whereas β-(1,6)-glucan represents 10–15% of the total yeast cell wall polysaccharides, each chain of the latter extending up to 140–350 glucose residues in length. The amount of 3,6-branched glucose residues varies with the yeast species: 7, 15, and 75% in S. cerevisiae, Candida albicans, and Schizosaccharomyces pombe, respectively (1). β-(1,6)-Glucan stabilizes the cell wall, since it plays a central role as a linker for specific cell wall components, including β-(1,3)-glucan, chitin, and mannoproteins (2, 3). However, the exact structure of the β-(1,6)-glucan and the mode of biosynthesis of this polymer are largely unknown. In S. pombe, immunodetection studies suggested that synthesis of this polymer backbone begins in the endoplasmic reticulum, with extension occurring in the Golgi (4) and final processing at the plasma membrane. In S. cerevisiae, Montijn and co-workers (5), by immunogold labeling, detected β-(1,6)-glucan at the plasma membrane, suggesting that the synthesis takes place largely at the cell surface.More than 20 genes, including the KRE gene family (14 members) and their homologues, SKN1 and KNH1, have been reported to be involved in β-(1,6)-glucan synthesis in S. cerevisiae, C. albicans, and Candida glabrata (610). Among all of these genes, the ones that seem to play the major synthetic role are KRE5 and KRE9, since their disruption caused significant reduction (100 and 80%, respectively, relative to wild type) in the cell wall β-(1,6)-glucan content (1113).To date, the biochemical reaction responsible for the synthesis of β-(1,6)-glucan and the product synthesized remained unknown. Indeed, in most cases, when membrane preparations are incubated with UDP-glucose, only linear β-(1,3)-glucan polymers are produced, although some studies have reported the production of low amounts of β-(1,6)-glucans by membrane preparations (1417). These data suggest that disruption of the fungal cell prevents or at least has a strong negative effect on β-(1,6)-glucan synthesis. The use of permeabilized cells, which allows substrates, such as nucleotide sugar precursors, to be readily transported across the plasma membrane, is an alternative method to study in situ cell wall enzyme activities (1822). A number of methods have been developed to permeabilize the yeast cell wall (23), of which osmotic shock was successfully used to demonstrate β-(1,3)-glucan and chitin synthase activities (20, 24). Herein, we describe the biochemical activity responsible for β-(1,6)-glucan synthesis using permeabilized S. cerevisiae cells and UDP-[14C]glucose as a substrate. We also have analyzed the physicochemical parameters of this activity and chemically characterized the end product and its structural organization within the mature yeast cell wall.  相似文献   

11.
Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in ∼95% of cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis. A structure of the bovine enzyme that we published previously (Zhao, B., Lei, L., Kagawa, N., Sundaramoorthy, M., Banerjee, S., Nagy, L. D., Guengerich, F. P., and Waterman, M. R. (2012) Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants. J. Biol. Chem. 287, 10613–10622), containing two molecules of the substrate 17α-hydroxyprogesterone, has been used as a template for understanding genetic deficiencies. We have now obtained a crystal structure of human P450 21A2 in complex with progesterone, a substrate in adrenal 21-hydroxylation. Substrate binding and release were fast for human P450 21A2 with both substrates, and pre-steady-state kinetics showed a partial burst but only with progesterone as substrate and not 17α-hydroxyprogesterone. High intermolecular non-competitive kinetic deuterium isotope effects on both kcat and kcat/Km, from 5 to 11, were observed with both substrates, indicative of rate-limiting C–H bond cleavage and suggesting that the juxtaposition of the C21 carbon in the active site is critical for efficient oxidation. The estimated rate of binding of the substrate progesterone (kon 2.4 × 107 m−1 s−1) is only ∼2-fold greater than the catalytic efficiency (kcat/Km = 1.3 × 107 m−1 s−1) with this substrate, suggesting that the rate of substrate binding may also be partially rate-limiting. The structure of the human P450 21A2-substrate complex provides direct insight into mechanistic effects of genetic variants.  相似文献   

12.
《Carbohydrate research》1988,172(1):1-10
Interaction of β-d-fructose with hydrated salts of zinc-group-metal has been studied in aqueous solution and solid adducts of the type M(d-fructose)X2·nH2O, where M = Zn(II), Cd(II), and Hg(II) ions, X = Cl or Br, and n = 0–2, have been isolated, and characterized by means of F.t.-i.r. spectroscopy, X-ray powder diffraction, and molar conductivity measurements. The marked spectral similarities observed with the Mg(d-fructose)X2·4 H2O (X = Cl or Br) compounds indicated that the Zn(II) and Cd(II) ions are six-coordinated, binding to two d-fructose molecules through O-2, O-3 of the first d-fructose, and O-4, O-5 of the second, as well as to two H2O. The Hg(II) ion binds to two sugar moieties in the same fashion as do the Zn(II) and Cd(II) ions, resulting in four-coordination geometry around the mercury ion. The crystalline sugar is in the β-d-fructopyranose form, and the coordination of the of the Ca(II) ion takes place through the β-d-fructopyranose isomer, whereas the binding of the Mg(II), Zn(II), Cd(II), Hg(II), and UO2+2 cations could be via the β-d-fructopyranose and the β-d-fructofuranose structures.  相似文献   

13.
Systemic amyloidosis is a fatal disease caused by misfolding of native globular proteins, which then aggregate extracellularly as insoluble fibrils, damaging the structure and function of affected organs. The formation of amyloid fibrils in vivo is poorly understood. We recently identified the first naturally occurring structural variant, D76N, of human β2-microglobulin (β2m), the ubiquitous light chain of class I major histocompatibility antigens, as the amyloid fibril protein in a family with a new phenotype of late onset fatal hereditary systemic amyloidosis. Here we show that, uniquely, D76N β2m readily forms amyloid fibrils in vitro under physiological extracellular conditions. The globular native fold transition to the fibrillar state is primed by exposure to a hydrophobic-hydrophilic interface under physiological intensity shear flow. Wild type β2m is recruited by the variant into amyloid fibrils in vitro but is absent from amyloid deposited in vivo. This may be because, as we show here, such recruitment is inhibited by chaperone activity. Our results suggest general mechanistic principles of in vivo amyloid fibrillogenesis by globular proteins, a previously obscure process. Elucidation of this crucial causative event in clinical amyloidosis should also help to explain the hitherto mysterious timing and location of amyloid deposition.  相似文献   

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1. Coptotermes guangzhouensis, sp. n. This new sepcies can be pesarated from C.formosanus Shiraki, by thelength of left mandble<0.95, by the posterior margin of pronotum almoststraight, and by the width of head 0.99-1.12 (1.06).  相似文献   

16.
During DNA repair, DNA polymerase β (Pol β) is a highly dynamic enzyme that is able to select the correct nucleotide opposite a templating base from a pool of four different deoxynucleoside triphosphates (dNTPs). To gain insight into nucleotide selection, we use a fluorescence resonance energy transfer (FRET)-based system to monitor movement of the Pol β fingers domain during catalysis in the presence of either correct or incorrect dNTPs. By labeling the fingers domain with ((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS) and the DNA substrate with Dabcyl, we are able to observe rapid fingers closing in the presence of correct dNTPs as the IAEDANS comes into contact with a Dabcyl-labeled, one-base gapped DNA. Our findings show that not only do the fingers close after binding to the correct dNTP, but that there is a second conformational change associated with a non-covalent step not previously reported for Pol β. Further analyses suggest that this conformational change corresponds to the binding of the catalytic metal into the polymerase active site. FRET studies with incorrect dNTP result in no changes in fluorescence, indicating that the fingers do not close in the presence of incorrect dNTP. Together, our results show that nucleotide selection initially occurs in an open fingers conformation and that the catalytic pathways of correct and incorrect dNTPs differ from each other. Overall, this study provides new insight into the mechanism of substrate choice by a polymerase that plays a critical role in maintaining genome stability.  相似文献   

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The entire skeleton ofTephrinectes sinensis, the single representative of a monotypic genus, is described in detail. The apomorphic characters observed suggest that the sister group ofT. sinensis is a clade composed of the Poecilopsettinae, Rhombosoleinae, Samarinae, Achiridae, Soleidae and Cynoglossidae, taxa which share an anteriorly-inclined second neural spine the distal portion of which overlies the cranium. This supports the removal ofT. sinensis from its former position in the Paralichthyidae.  相似文献   

20.
The present study re-examines the detailed morphology of the type-species, Diclidophora merlangi (Kuhn, in Nordmann, 1832) Krøyer, 1838, and other Diclidophora species parasitic on gadid fishes: D. denticulata (Olsson, 1876) Price, 1943, D. esmarkii (Th. Scott, 1901) Sproston, 1946, D. luscae (van Beneden & Hesse, 1863) Price, 1943, D. minor (Olsson, 1868) Sproston, 1946, D. palmata (Leuckart, 1830) Diesing, 1850, D. phycidis (Parona & Perugia, 1889) Sproston, 1946, D. pollachii (van Beneden & Hesse, 1863) Price, 1943 and the recently described D. micromesisti Suriano & Martorelli, 1984. An amended generic diagnosis of Diclidophora Krøyer, 1838 (synonym Diclidophora Diesing, 1850) is provided, which includes the presence of a prostatic vesicle in the terminal male genitalia and the distal fusion of the median and peripheral sclerites, a1 and c1 in the clamp anterior jaw. Macrouridophora n. g. is herein proposed for species previously considered in Diclidophora, which are parasitic on macrourid and morid fishes. The clamp morphology in Macrouridophora n. g. has distinct lamellate extension attachments to peripheral sclerites c1 and the distal portion of d1, with no distal fusion between a1 and c1 in the anterior jaw. Macrouridophora macruri (Brinkmann, 1942) n. comb. is chosen as the type-species. Nine other species are herein transferred to Macrouridophora n. g.: M. coelorhynchi (Robinson, 1961) n. comb., M. lotella (Machida, 1972) n. comb., M. nezumiae (Munroe, Campbell & Zwerner, 1981) n. comb. and M. tubiformis (Rohde & Williams, 1987) n. comb. are redescribed, based on the re-examination of type or voucher specimens. Macrouridophora attenuata (Mamaev & Zubtschenko, 1979) n. comb., M. caudata (Mamaev & Zubtschenko, 1984) n. comb., M. papilio (Mamaev & Avdeev, 1981) n. comb., M. paracoelorhynchi (Mamaev & Paruchin, 1979) n. comb. and M. physiculi (Mamaev & Avdeev, 1981) n. comb. have adequately described haptoral clamps in the literature. The clamp morphology in Macrouridophora sp. from Lepidorhynchus denticulatus in Australia is also considered. Diclidophora whitsonii Suriano & Martorelli, 1984 is herein transferred to the genus Macruricotyle Mamaev & Ljadov, 1975, as M. whitsonii (Suriano & Martorelli, 1984) n. comb. D. embiotocae Hanson, 1979 is herein considered a species incertae sedis. D. caudospina Khan & Karyakarte, 1983 and D. paddiforma Deo & Karyakarte, 1979 are herein considered species inquirendae. D. aglandulosa Deo, 1977, D. glandulosa Das, 1972, D. minuta Das, 1972 and D. spindale Deo, 1977 are formally dismissed as nomina nuda. The systematic position of Diclidophora Krøyer, 1838 and Macrouridophora n. g. in the subfamily Diclidophorinae Cerfontaine, 1895 (sensu Mamaev, 1976) is discussed.  相似文献   

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