scholarly journals Characterization of an Endo-β-1,6-Galactanase from Streptomyces avermitilis NBRC14893

2008 ◽  
Vol 74 (8) ◽  
pp. 2379-2383 ◽  
Author(s):  
Hitomi Ichinose ◽  
Toshihisa Kotake ◽  
Yoichi Tsumuraya ◽  
Satoshi Kaneko
Keyword(s):  
Signal Sequence ◽  
Recombinant Enzyme ◽  
Streptomyces Avermitilis ◽  
Glycoside Hydrolase Family ◽  
Reading Frame ◽  
Secretion Signal ◽  
Acid Protein ◽  
Hydrolysis Of ◽  
Amino Acid Protein

ABSTRACT The putative endo-β-1,6-galactanase gene from Streptomyces avermitilis was cloned and expressed in Escherichia coli, and the enzymatic properties of the recombinant enzyme were characterized. The gene consisted of a 1,476-bp open reading frame and encoded a 491-amino-acid protein, comprising an N-terminal secretion signal sequence and glycoside hydrolase family 5 catalytic module. The recombinant enzyme, Sa1,6Gal5A, catalyzed the hydrolysis of β-1,6-linked galactosyl linkages of oligosaccharides and polysaccharides. The enzyme produced galactose and a range of β-1,6-linked galacto-oligosaccharides, predominantly β-1,6-galactobiose, from β-1,6-galactan chains. There was a synergistic effect between the enzyme and Sa1,3Gal43A in degrading tomato arabinogalactan proteins. These results suggest that Sa1,6Gal5A is the first identified endo-β-1,6-galactanase from a prokaryote.

Cloning and characterization of a 2,3-oxidosqualene cyclase from Eleutherococcus trifoliatus

Holzforschung ◽  
2013 ◽  
Vol 67 (4) ◽  
pp. 463-471 ◽  
Author(s):  
Li-Ting Ma ◽  
Sheng-Yang Wang ◽  
Yen-Hsueh Tseng ◽  
Yi-Ru Lee ◽  
Fang-Hua Chu
Keyword(s):  
Physiological Role ◽  
Active Site Residues ◽  
Reading Frame ◽  
Oxidosqualene Cyclases ◽  
Oxidosqualene Cyclase ◽  
Acid Protein ◽  
Leaf Tissues ◽  
New Perspective ◽  
Amino Acid Protein

Abstract The 2,3-oxidosqualene cyclases (OSCs) are a family of enzymes that have an important role in plant triterpene biosynthesis. In this study, an OSC gene designed EtLUS from Eleutherococcus trifoliatus has been cloned. EtLUS includes a 2292-bp open reading frame and encodes a 763-amino acid protein. EtLUS has an MLCYCR motif, which is conserved in lupeol synthases. Comparison of active-site residues and gene expression in yeast showed that EtLUS synthesizes lupeol. However, EtLUS has the highest sequence identity with β-amyrin synthases from Araliaceae rather than lupeol synthases, adding new perspective to the evolution of the OSCs of Araliaceae. Furthermore, EtLUS is upregulated in leaf tissues under methyl jasmonate treatment, which can be interpreted that lupeol and its derivatives play an ecological and physiological role in plant defense against pathogens and insect herbivores.

scholarly journals Identification and characterization of a cDNA encoding mouse CAP: a homolog of the yeast adenylyl cyclase associated protein

1993 ◽  
Vol 105 (3) ◽  
pp. 777-785 ◽  
Author(s):  
A.B. Vojtek ◽  
J.A. Cooper
Keyword(s):  
Adenylyl Cyclase ◽  
Leading Edge ◽  
Northern Analysis ◽  
Reading Frame ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Acid Protein ◽  
Carboxy Terminal ◽  
Amino Acid Protein

CAP, an adenylyl cyclase associated protein, is present in Saccharomyces cerevisiae and Schizosaccharomyces pombe. In both organisms, CAP is bifunctional: the N-terminal domain binds to adenylyl cyclase, thereby enabling adenylyl cyclase to respond appropriately to upstream regulatory signals, such as RAS in S. cerevisiae; the C-terminal domain is required for cellular morphogenesis. Here, we describe the isolation of a cDNA encoding a CAP homolog from a higher eukaryote. The mouse CAP cDNA contains an open reading frame capable of encoding a 474 amino acid protein. The protein encoded by the mouse CAP cDNA shows extensive homology to the yeast CAP proteins, particularly in the central poly-proline rich region and in the C-terminal domain. By northern analysis, the CAP message appears to be ubiquitous, but not uniform. By indirect immunofluorescence, ectopically expressed mouse CAP protein is found in the cytoplasm of fibroblasts and, in migrating cells, at the leading edge. Expression of the mouse CAP cDNA in S. cerevisiae complements defects associated with loss of the yeast CAP carboxy-terminal domain. Hence, the function of the CAP carboxy-terminal domain has been conserved from yeast to mouse.

scholarly journals Molecular Cloning and Characterization of a cDNA Encoding Sedoheptulose-1,7-bisphosphatase from Mulberry (Morus alba var. multicaulis)

2008 ◽  
Vol 57 (1-6) ◽  
pp. 152-157 ◽  
Author(s):  
X. Ji ◽  
Y. Gai ◽  
J. Ma ◽  
C. Zheng ◽  
Z. Mu
Keyword(s):  
Morus Alba ◽  
Evolutionary Analysis ◽  
Comparison Analysis ◽  
Reading Frame ◽  
Acid Protein ◽  
Fructose 1,6 Bisphosphatase ◽  
Rapid Amplification ◽  
Molecular Evolutionary Analysis ◽  
Amino Acid Protein

Abstract A full-length cDNA encoding sedoheptulose-1,7-bisphosphatase (SBPase; EC 3.1.3.37) was cloned from mulberry (Morus alba var. multicaulis) by rapid amplification of cDNA ends (RACE). The cDNA consisted of 1,527 nucleotides with an open reading frame (ORF) of 1,179 nucleotides encoding a 393 amino acid protein of approximately 42.6 kDa. Sequence comparison analysis showed that mulberry SBPase (MSBPase) had high homology to other plant counterparts. Phylogenetic and molecular evolutionary analysis revealed that MSBPase fell into plant SBPase group. Moreover, SBPase and fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) shared 28-32% identical residues, suggesting that the two enzymes originated from the same evolution branch. Molecular modeling indicated that each subunit of MSBPase was composed of α-helices and β-sheets joined by turns and loops, and folded into a structure of hexahedron shape which was very similar to FBPase.

scholarly journals Molecular Cloning and Biochemical Characterization of a Recombinant Sterol 3-O-Glucosyltransferase fromGymnema sylvestreR.Br. Catalyzing Biosynthesis of Steryl Glucosides

2014 ◽  
Vol 2014 ◽  
pp. 1-14 ◽  
Author(s):  
Pragya Tiwari ◽  
Rajender Singh Sangwan ◽  
Asha ◽  
B. N. Mishra ◽  
Farzana Sabir ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Present Report ◽  
Hydroxyl Group ◽  
Biological Origin ◽  
Reading Frame ◽  
Steryl Glucosides ◽  
Acid Protein ◽  
Phytochemical Profiles ◽  
Amino Acid Protein

Gymnema sylvestreR.Br., a pharmacologically important herb vernacularly called Gur-Mar (sugar eliminator), is widely known for its antidiabetic action. This property of the herb has been attributed to the presence of bioactive triterpene glycosides. Although some information regarding pharmacology and phytochemical profiles of the plant are available, no attempts have been made so far to decipher the biosynthetic pathway and key enzymes involved in biosynthesis of steryl glucosides. The present report deals with the identification and catalytic characterization of a glucosyltransferase, catalyzing biosynthesis of steryl glycosides. The full length cDNA (2572 bp) contained an open reading frame of 2106 nucleotides that encoded a 701 amino acid protein, falling into GT-B subfamily of glycosyltransferases. The GsSGT was expressed inEscherichia coliand biochemical characterization of the recombinant enzyme suggested its key role in the biosynthesis of steryl glucosides with catalytic preference for C-3 hydroxyl group of sterols. To our knowledge, this pertains to be the first report on cloning and biochemical characterization of a sterol metabolism gene fromG. sylvestreR.Br. catalyzing glucosylation of a variety of sterols of biological origin from diverse organisms such as bacteria, fungi, and plants.

Isolation and characterization of the muscle-specific isoform of creatine kinase from the zebrafish, Danio rerio

2001 ◽  
Vol 79 (6) ◽  
pp. 779-782 ◽  
Author(s):  
Gregory Harder ◽  
Ross McGowan
Keyword(s):  
Creatine Kinase ◽  
Amino Acid ◽  
Danio Rerio ◽  
Cdna Sequence ◽  
Reading Frame ◽  
Isolation And Characterization ◽  
Acid Protein ◽  
Amino Acid Levels ◽  
Amino Acid Protein

We have isolated and characterized a cDNA sequence corresponding to the zebrafish muscle-specific isoform of creatine kinase. The sequence is 1552 bases in length and contains an open reading frame capable of producing a 381 amino acid protein. The sequence is very similar to muscle-specific creatine kinases isolated from other species at both the nucleotide and amino acid levels but contains some differences from a previously reported zebrafish clone.Key words: creatine kinase, muscle isoform, zebrafish, Danio rerio.

scholarly journals Characterization of an Unusual Cold-Active β-Glucosidase Belonging to Family 3 of the Glycoside Hydrolases from the Psychrophilic Isolate Paenibacillus sp. Strain C7

2005 ◽  
Vol 71 (8) ◽  
pp. 4225-4232 ◽  
Author(s):  
Stephanie Shipkowski ◽  
Jean E. Brenchley
Keyword(s):  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Galactosidase Activity ◽  
Reading Frame ◽  
Substrate Preferences ◽  
Acid Protein ◽  
Paenibacillus Sp ◽  
Cold Active ◽  
Ph Range

ABSTRACT We selected for spore-forming psychrophilic bacteria able to use lactose as a carbon source and one isolate, designated Paenibacillus sp. strain C7, that was phylogenetically related to, but distinct from both Paenibacillus macquariensis and Paenibacillus antarcticus. Some Escherichia coli transformants obtained with genomic DNA from this isolate hydrolyzed X-Gal (5-bromo-4-chloro-3-indoyl-β-d-galactopyranoside) only below 30°C, an indication of cold-active β-galactosidase activity. Sequencing of the cloned insert revealed an open reading frame encoding a 756-amino acid protein that, rather than belonging to a family typically known for β-galactosidase activity, belonged to glycoside hydrolase family 3, a family of β-glucosidases. Because of this unusual placement, the recombinant enzyme (BglY) was purified and characterized. Consistent with its classification, the enzyme had seven times greater activity with the glucoside substrate ONPGlu (o-nitrophenyl-β-d-glucopyranoside) than with the galactoside substrate ONPGal (o-nitrophenyl-β-d-galactopyranoside). In addition, the enzyme had, with ONPGlu, a thermal optimum around 30 to 35°C, activity over a broad pH range (5.5 to 10.9), and an especially low Km (<0.003 mM). Further examination of substrate preference showed that the BglY enzyme also hydrolyzed other aryl-β-glucosides such as helicin, MUG (4-methylumbelliferyl-β-d-glucopyranoside), esculin, indoxyl-β-d-glucoside (a natural indigo precursor), and salicin, but had no activity with glucosidic disaccharides or lactose. These characteristics and substrate preferences make the BglY enzyme unique among the family 3 β-glucosidases. The hydrolysis of a variety of aryl-β-glucosides suggests that the enzyme may allow the organism to use these substrates in the environment and that its low Km on indoxyl-β-d-glucoside may make it useful for producing indigo.

scholarly journals Molecular cloning and characterization of a novel β-1,3-xylanase possessing two putative carbohydrate-binding modules from a marine bacterium Vibrio sp. strain AX-4

2005 ◽  
Vol 388 (3) ◽  
pp. 949-957 ◽  
Author(s):  
Masashi KIYOHARA ◽  
Keishi SAKAGUCHI ◽  
Kuniko YAMAGUCHI ◽  
Toshiyoshi ARAKI ◽  
Takashi NAKAMURA ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Marine Bacterium ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Carbohydrate Binding Modules ◽  
Hydrolysis Of ◽  
Hydrolase Family

We cloned a novel β-1,3-xylanase gene, consisting of a 1728-bp open reading frame encoding 576 amino acid residues, from a marine bacterium, Vibrio sp. strain AX-4. Sequence analysis revealed that the β-1,3-xylanase is a modular enzyme composed of a putative catalytic module belonging to glycoside hydrolase family 26 and two putative carbohydrate-binding modules belonging to family 31. The recombinant enzyme hydrolysed β-1,3-xylan to yield xylo-oligosaccharides with different numbers of xylose units, mainly xylobiose, xylotriose and xylotetraose. However, the enzyme did not hydrolyse β-1,4-xylan, β-1,4-mannan, β-1,4-glucan, β-1,3-xylobiose or p-nitrophenyl-β-xyloside. When β-1,3-xylo-oligosaccharides were used as the substrate, the kcat value of the enzyme for xylopentaose was found to be 40 times higher than that for xylotetraose, and xylotriose was extremely resistant to hydrolysis by the enzyme. A PSI-BLAST search revealed two possible catalytic Glu residues (Glu-138 as an acid/base catalyst and Glu-234 as a nucleophile), both of which are generally conserved in glycoside hydrolase superfamily A. Replacement of these two conserved Glu residues with Asp and Gln resulted in a significant decrease and complete loss of enzyme activity respectively, without a change in their CD spectra, suggesting that these Glu residues are the catalytic residues of β-1,3-xylanase. The present study also clearly shows that the non-catalytic putative carbohydrate-binding modules play an important role in the hydrolysis of insoluble β-1,3-xylan, but not that of soluble glycol-β-1,3-xylan. Furthermore, repeating a putative carbohydrate-binding module strongly enhanced the hydrolysis of the insoluble substrate.

scholarly journals Knockdown of β-N-acetylglucosaminidase 2 Impairs Molting and Wing Development in Lasioderma serricorne (Fabricius)

Insects ◽  
2019 ◽  
Vol 10 (11) ◽  
pp. 396 ◽  
Author(s):  
Wen-Jia Yang ◽  
Kang-Kang Xu ◽  
Xin Yan ◽  
Can Li
Keyword(s):  
Developmental Stages ◽  
Catalytic Domain ◽  
Glycoside Hydrolase Family ◽  
Wing Development ◽  
Lasioderma Serricorne ◽  
Chitin Synthesis ◽  
Reading Frame ◽  
Acid Protein ◽  
Carbohydrate Enzymes ◽  
Amino Acid Protein

β-N-acetylglucosaminidases (NAGs) are carbohydrate enzymes that degrade chitin oligosaccharides into N-acetylglucosamine monomers. This process is important for chitin degradation during insect development and metamorphosis. We identified and evaluated a β-N-acetylglucosaminidase 2 gene (LsNAG2) from the cigarette beetle, Lasioderma serricorne (Fabricius). The full-length open reading frame of LsNAG2 was 1776 bp and encoded a 591 amino acid protein. The glycoside hydrolase family 20 (GH20) catalytic domain and an additional GH20b domain of the LsNAG2 protein were highly conserved. Phylogenetic analysis revealed that LsNAG2 clustered with the group II NAGs. Quantitative real-time PCR analyses showed that LsNAG2 was expressed in all developmental stages and was most highly expressed in the late larval and late pupal stages. In the larval stage, LsNAG2 was predominantly expressed in the integument. Knockdown of LsNAG2 in fifth instar larvae disrupted larval–pupal molting and reduced the expression of four chitin synthesis genes (trehalase 1 (LsTRE1), UDP-N-acetylglucosamine pyrophosphorylase 1 and 2 (LsUAP1 and LsUAP2), and chitin synthase 1 (LsCHS1)). In late pupae, LsNAG2 depletion resulted in abnormal adult eclosion and wing deformities. The expression of five wing development-related genes (teashirt (LsTSH), vestigial (LsVG), wingless (LsWG), ventral veins lacking (LsVVL), and distal-less (LsDLL)) significantly declined in the LsNAG2-depleted beetles. These findings suggest that LsNAG2 is important for successful molting and wing development of L. serricorne.

scholarly journals Molecular Characterization of DSR-E, an α-1,2 Linkage-Synthesizing Dextransucrase with Two Catalytic Domains

2002 ◽  
Vol 184 (20) ◽  
pp. 5753-5761 ◽  
Author(s):  
Sophie Bozonnet ◽  
Marguerite Dols-Laffargue ◽  
Emeline Fabre ◽  
Sandra Pizzut ◽  
Magali Remaud-Simeon ◽  
...  
Keyword(s):  
Leuconostoc Mesenteroides ◽  
Catalytic Mechanism ◽  
Catalytic Domain ◽  
Carboxy Terminus ◽  
Reading Frame ◽  
Average Mass ◽  
Catalytic Domains ◽  
Acid Protein ◽  
Amino Acid Protein

ABSTRACT A novel Leuconostoc mesenteroides NRRL B-1299 dextransucrase gene, dsrE, was isolated, sequenced, and cloned in Escherichia coli, and the recombinant enzyme was shown to be an original glucansucrase which catalyses the synthesis of α-1,6 and α-1,2 linkages. The nucleotide sequence of the dsrE gene consists of an open reading frame of 8,508 bp coding for a 2,835-amino-acid protein with a molecular mass of 313,267 Da. This is twice the average mass of the glucosyltransferases (GTFs) known so far, which is consistent with the presence of an additional catalytic domain located at the carboxy terminus of the protein and of a central glucan-binding domain, which is also significantly longer than in other glucansucrases. From sequence comparison with family 70 and α-amylase enzymes, crucial amino acids involved in the catalytic mechanism were identified, and several original sequences located at some highly conserved regions in GTFs were observed in the second catalytic domain.

scholarly journals Characterization of the SSAT1 gene and its expression profiling in various tissues and follicles in geese

2018 ◽  
Vol 18 (3) ◽  
pp. 675-684
Author(s):  
Dongmei Jiang ◽  
Ziyu Chen ◽  
Zhixin Yi ◽  
Bo Kang
Keyword(s):  
Pineal Gland ◽  
Mrna Expression ◽  
Expression Profiles ◽  
Reading Frame ◽  
Cell Proliferation And Differentiation ◽  
Acid Protein ◽  
Preovulatory Follicles ◽  
Proliferation And Differentiation ◽  
Amino Acid Protein

Abstract Spermidine/spermine N1-acetyltransferase (SSAT ) is a catabolic regulator of polyamines, ubiquitous molecules essential for cell proliferation and differentiation. In this study, the molecular characterization of the SSAT1 gene of the Sichuan white goose was analyzed, as well as its expression profiles in various follicles and tissues. The open reading frame of the SSAT1 cDNA (GenBank No. KM925008) is 516 bp in length and encodes a 171-amino acid protein with a putative molecular weight of 20 kDa. The predicted SSAT1 protein is highly conserved with those of other species, especially Gallus gallus. SSAT1 mRNA was ubiquitously expressed in all the examined tissues. The highest level of SSAT1 mRNA expression was found in the pineal gland (P<0.05), and was 12-fold greater than in the heart. The level of SSAT1 mRNA expression was relatively lower in preovulatory follicles, while it was higher in postovulatory follicles (POFs), particularly in POF1. Furthermore, as postovulatory follicles degenerated, SSAT1 expression gradually decreased. Our findings suggest that SSAT1 might play important roles in mediating the physiological function of the pineal gland and regulating the regression of POFs.

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