scholarly journals Protein structure and gene cloning of Syncephalastrum racemosum nuclease

1999 ◽  
Vol 339 (2) ◽  
pp. 261-267 ◽  
Author(s):  
Heng-Chien HO ◽  
Ta-Hsiu LIAO
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Direct Sequencing ◽  
Coli Strain ◽  
Protein Sequencing ◽  
Intact Protein ◽  
Computer Search ◽  
Degenerate Primers ◽  
Syncephalastrum Racemosum

The complete amino acid sequence of the fungus Syncephalastrum racemosum (Sr-) nuclease has been delineated on the basis of protein sequencing of the intact protein and its protease-digested peptides. The resulting 250-residue sequence shows a carbohydrate side chain attached at Asn134 and two half-cystine residues (Cys242 and Cys247) cross-linked to form a small disulphide loop. On the basis of the sequence of Sr-nuclease, a computer search in the sequence database yielded 60% and 48% positional identities with the sequences of Cunninghamella echinulata nuclease C1 and yeast mitochondria nuclease respectively, and very little similarity to those of several known mammalian DNases I. Sequence alignment of the three similar nucleases reveals that the single small disulphide loop is unchanged but the carbohydrate attachment in Sr-nuclease is absent from the other two nucleases. Alignment also shows a highly conserved region harbouring Sr-nuclease His85, which is assigned as one of the essential residues in the active site. The cDNA encoding Sr-nuclease was amplified by using reverse transcriptase-mediated PCR with degenerate primers based on its amino acid sequence. Subsequently, specific primers were synthesized for use in the 3´ and 5´ rapid amplification of cDNA ends (RACE). Direct sequencing of the RACE products led to the deduction of a 1.1 kb cDNA sequence for Sr-nuclease. The cDNA contains an open reading frame of 320 amino acid residues including a 70-residue putative signal peptide and the 250-residue mature protein. Finally, the recombinant Sr-nuclease was expressed in Escherichia coli strain BL21(DE3) in which the recombinant protein, after solubilization with detergent and renaturation, showed both DNase and RNase activities. The assignment of His85 to the active site was further supported by evidence that the mutant protein Sr-nuclease (H85A), in which His85 was replaced by Ala, was not able to degrade DNA or RNA.

scholarly journals Cloning and characterization of a novel nuclease from shrimp hepatopancreas, and prediction of its active site

2000 ◽  
Vol 346 (3) ◽  
pp. 799-804 ◽  
Author(s):  
Wen-Yi WANG ◽  
Shwu-Huey LIAW ◽  
Ta-Hsiu LIAO
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Sequence Similarity ◽  
Distinct Group ◽  
Structural Data ◽  
Hydrolytic Activity ◽  
Degenerate Primers ◽  
Homologous Proteins ◽  
Reading Frame

Approximately 95% of the amino acid sequence of a shrimp (Penaeus japonicus) nuclease was derived from protease-digested peptides. A 1461-base cDNA for the nuclease was amplified and sequenced with degenerate primers based on the amino acid sequence and then specific primers by 3ʹ and 5ʹ RACE (rapid amplification of cDNA ends). It contains an open reading frame encoding a putative 21-residue signal peptide and a 381-residue mature protein. The N-terminus of the enzyme is pyroglutamate, deduced from composition and matrix-assisted laser desorption ionization-time-of-flight MS analyses, and confirmed by a glutamine residue in the cDNA sequence. The enzyme has 11 Cys residues, forming five intramolecular disulphides. The eleventh Cys residue was linked to a thiol compound with an estimated molecular mass of between 500 and 700 Da. A sequence similarity search revealed no homologous proteins but residues 205-255 shared a conserved active-site motif within a distinct group of nucleases. His211 in this conserved motif was shown to be very important in catalysis by site-specific modification with 14C-labelled iodoacetate. The shrimp nuclease, previously designated DNase I, does indeed possess a low level of hydrolytic activity towards RNA in the presence of Mg2+ and Ca2+. The conservation of functionally important residues during distant evolution might imply that the catalytic mechanisms are similar in these nucleases, which should be classified in one subfamily. Finally, an active-site structure for shrimp nuclease was proposed on the basis of published structural data and the results of mutational and biochemical analyses of Serratia nuclease.

scholarly journals Derived amino acid sequence and identification of active site residues of Escherichia coli beta-hydroxydecanoyl thioester dehydrase.

1988 ◽  
Vol 263 (10) ◽  
pp. 4641-4646 ◽  
Author(s):  
J E Cronan ◽  
W B Li ◽  
R Coleman ◽  
M Narasimhan ◽  
D de Mendoza ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Active Site Residues

scholarly journals Amino acid sequence of the active site of Acanthamoeba myosin II.

1986 ◽  
Vol 261 (4) ◽  
pp. 1844-1848
Author(s):  
M A Atkinson ◽  
E A Robinson ◽  
E Appella ◽  
E D Korn
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Myosin Ii

Glutamine active site of formylglycinamide ribonucleotide amidotransferase. 2. Amino acid sequence of labeled peptides

Biochemistry ◽  
1977 ◽  
Vol 16 (6) ◽  
pp. 1070-1076 ◽  
Author(s):  
Shiro Ohnoki ◽  
Bor-Shyue Hong ◽  
John M. Buchanan
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site

scholarly journals A New Hypothesis on the Strategy for Acquisition of Phosphorus in Arbuscular Mycorrhiza: Up-Regulation of Secreted Acid Phosphatase Gene in the Host Plant

2005 ◽  
Vol 18 (10) ◽  
pp. 1046-1053 ◽  
Author(s):  
Tatsuhiro Ezawa ◽  
Masahito Hayatsu ◽  
Masanori Saito
Keyword(s):  
Acid Phosphatase ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Mycorrhizal Colonization ◽  
Purple Acid Phosphatase ◽  
Terminal Amino Acid ◽  
Degenerate Primers ◽  
Fungal Partner ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

The mycorrhiza-responsive phosphatase of Tagetes patula in symbiosis with Glomus etunicatum was detected by electrophoresis, was purified by column chromatography, and was characterized as acid phosphatase that was secreted into rhizosphere. The N-terminal amino acid sequence was determined by a gas-phase sequencer, and a cDNA fragment of the phosphatase gene (TpPAP1) was amplified by degenerate primers designed based on the N-terminal amino acid sequence. The full-length cDNA was obtained by the rapid amplification of cDNA ends technique. The TpPAP1 was of host origin, and the cDNA was 1,843 bp long with a predicted open reading frame of polypeptide of 466 amino acids. Phylogenetic analysis revealed that the gene fell into the cluster of plant high-molecular-weight purple acid phosphatase. Expression analysis of the TpPAP1 in T. patula in symbiosis with Archaeospora leptoticha showed that the levels of transcripts increased eightfold by mycorrhizal colonization. Western blot analysis revealed that the 57-kDa protein corresponding to the mycorrhiza-responsive phosphatase increased by mycorrhizal colonization. The present study proposes a new strategy for acquisition of P in arbuscular mycorrhizal associations in which the fungal partner activates a part of the low-P adaptation system of the plant partner, phosphatase secretion, and improves the overall efficiency of P uptake.

scholarly journals Partial sequence of the purified protein confirms the identity of cDNA coding for human lysosomal α-mannosidase B

1995 ◽  
Vol 305 (2) ◽  
pp. 363-366 ◽  
Author(s):  
C Emiliani ◽  
S Martino ◽  
J L Stirling ◽  
B Maras ◽  
A Orlacchio
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Rapid Method ◽  
Protein Sequencing ◽  
Subunit Composition ◽  
Partial Sequence ◽  
Sequencing Analysis ◽  
Cloned Cdna

Human lysosomal alpha-mannosidase has been purified by a simple and rapid method in sufficient quantities for the analysis of its subunit composition and partial protein sequencing. Analysis of the N-terminal residues of the 30 kDa polypeptide has enabled us to confirm the identity of the recently cloned cDNA that was tentatively identified as that of lysosomal alpha-mannosidase [Nebes and Schmidt (1994) Biochem. Biophys. Res. Commun. 200, 239-245] and to locate the position of this polypeptide within the total deduced amino acid sequence. This finding will therefore provide a firm foundation for the characterization of alpha-mannosidosis mutations.

scholarly journals Amino acid sequence of an active site fragment from human diphosphoglycerate mutase

FEBS Letters ◽  
1980 ◽  
Vol 114 (1) ◽  
pp. 124-126 ◽  
Author(s):  
Neill W. Haggarty ◽  
Linda A. Fothergill
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site
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