A common precursor for the three subunits ofL-glutamate oxidase encoded bygoxgene fromStreptomyces platensisNTU3304

2001 ◽  
Vol 47 (3) ◽  
pp. 269-275 ◽  
Author(s):  
Chien-Yuan Chen ◽  
Wen-Tung Wu ◽  
Chang-Jen Huang ◽  
Mei-Huei Lin ◽  
Chen-Kai Chang ◽  
...  
Keyword(s):  
Amino Acid Sequences ◽  
Binding Motif ◽  
Protein Coding ◽  
Reading Frame ◽  
Streptomyces Platensis ◽  
C Terminus ◽  
Common Precursor ◽  
Glutamate Oxidase ◽  
Negative Effect ◽  
Molecular Masses

A segment of DNA containing the L-glutamate oxidase (gox) gene from Streptomyces platensis NTU3304 was cloned. The entire nucleotide sequence of the protein-coding portion consisting of 2130 bp (710 codons, including AUG and UGA) of the cloned DNA fragment was determined. The gox gene contained only one open reading frame (ORF) which coded for a 78-kDa polypeptide, the precursor of active extracellular Gox. Mature Gox is composed of three subunits, designated as α, β, and γ, with molecular masses of 39, 19, and 16 kDa, respectively. Analyses of the N-terminal amino acid sequences of the subunits revealed that the order of subunits in the precursor polypeptide encoded by the ORF, from N-terminus to C-terminus, is α–γ–β. The presence of the flavin adenine dinucleotide (FAD)-binding motif place Gox as a member of the flavoenzyme family. Furthermore, a negative effect of glucose on the biosynthesis of Gox was observed when it was used as carbon source.Key words: L-glutamate oxidase, gox gene, signal peptide, DNA sequence, flavoenzyme, pIJ702 vector.

scholarly journals Cloning and Characterization of Two Xyloglucanases from Paenibacillus sp. Strain KM21

2005 ◽  
Vol 71 (12) ◽  
pp. 7670-7678 ◽  
Author(s):  
Katsuro Yaoi ◽  
Tomonori Nakai ◽  
Yoshiro Kameda ◽  
Ayako Hiyoshi ◽  
Yasushi Mitsuishi
Keyword(s):  
Glycoside Hydrolase ◽  
Amino Acid Sequences ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
C Terminus ◽  
Paenibacillus Sp ◽  
Genes Encoding ◽  
Molecular Masses

ABSTRACT Two xyloglucan-specific endo-β-1,4-glucanases (xyloglucanases [XEGs]), XEG5 and XEG74, with molecular masses of 40 kDa and 105 kDa, respectively, were isolated from the gram-positive bacterium Paenibacillus sp. strain KM21, which degrades tamarind seed xyloglucan. The genes encoding these XEGs were cloned and sequenced. Based on their amino acid sequences, the catalytic domains of XEG5 and XEG74 were classified in the glycoside hydrolase families 5 and 74, respectively. XEG5 is the first xyloglucanase belonging to glycoside hydrolase family 5. XEG5 lacks a carbohydrate-binding module, while XEG74 has an X2 module and a family 3 type carbohydrate-binding module at its C terminus. The two XEGs were expressed in Escherichia coli, and recombinant forms of the enzymes were purified and characterized. Both XEGs had endoglucanase active only toward xyloglucan and not toward Avicel, carboxymethylcellulose, barley β-1,3/1,4-glucan, or xylan. XEG5 is a typical endo-type enzyme that randomly cleaves the xyloglucan main chain, while XEG74 has dual endo- and exo-mode activities or processive endo-mode activity. XEG5 digested the xyloglucan oligosaccharide XXXGXXXG to produce XXXG, whereas XEG74 digestion of XXXGXXXG resulted in XXX, XXXG, and GXXXG, suggesting that this enzyme cleaves the glycosidic bond of unbranched Glc residues. Analyses using various oligosaccharide structures revealed that unique structures of xyloglucan oligosaccharides can be prepared with XEG74.

scholarly journals Structure of transcripts from the homeotic Antennapedia gene of Drosophila melanogaster: two promoters control the major protein-coding region.

1986 ◽  
Vol 6 (12) ◽  
pp. 4676-4689 ◽  
Author(s):  
A Laughon ◽  
A M Boulet ◽  
J R Bermingham ◽  
R A Laymon ◽  
M P Scott
Keyword(s):  
Drosophila Melanogaster ◽  
Alternative Polyadenylation ◽  
Transcription Unit ◽  
Cdna Clones ◽  
Major Protein ◽  
Coding Region ◽  
Developmentally Regulated ◽  
Protein Coding ◽  
Reading Frame ◽  
C Terminus

The Antennapedia (Antp) homeotic gene of Drosophila melanogaster regulates segmental identity in the thorax. Loss of Antp function results in altered development of the embryonic thoracic segments or can cause legs to be transformed into antennae. Certain combinations of Antp recessive lethal alleles complement to permit normal development. The structure of the Antp gene, analyzed by sequencing cDNA clones and exons and by transcript mapping, revealed some of the basis for its genetic complexity. It has two promoters governing two nested transcription units, one unit 36 and one 103 kilobase pairs (kb) long. Both units incorporated the same protein-coding exons, all of which are located in the 3'-most 13 kb of the gene. The two promoters resulted in the attachment of either of two long noncoding leader sequences (1.5 and 1.7 kb) to a 1.1-kb open reading frame. Both transcription units used the same pair of alternative polyadenylation sites 1.4 kb apart; the choice of sites was developmentally regulated. Some of the mutations that disrupt the larger transcription unit complemented a mutation affecting the smaller one. Dominant mutations that transform antennae into legs split the gene but left the coding exons intact. The encoded protein has unusually long runs of glutamine and a homeodomain near the C terminus.

scholarly journals A common precursor for the three subunits of L-glutamate oxidase encoded by gox gene from Streptomyces platensis NTU3304

2001 ◽  
Vol 47 (3) ◽  
pp. 269-275 ◽  
Author(s):  
Chien-Yuan Chen ◽  
Wen-Tung Wu ◽  
Chang-Jen Huang ◽  
Mei-Huei Lin ◽  
Chen-Kai Chang ◽  
...  
Keyword(s):  
Streptomyces Platensis ◽  
Common Precursor ◽  
Glutamate Oxidase ◽  
I Gene

Structure of transcripts from the homeotic Antennapedia gene of Drosophila melanogaster: two promoters control the major protein-coding region

1986 ◽  
Vol 6 (12) ◽  
pp. 4676-4689
Author(s):  
A Laughon ◽  
A M Boulet ◽  
J R Bermingham ◽  
R A Laymon ◽  
M P Scott
Keyword(s):  
Drosophila Melanogaster ◽  
Alternative Polyadenylation ◽  
Transcription Unit ◽  
Cdna Clones ◽  
Major Protein ◽  
Coding Region ◽  
Developmentally Regulated ◽  
Protein Coding ◽  
Reading Frame ◽  
C Terminus

The Antennapedia (Antp) homeotic gene of Drosophila melanogaster regulates segmental identity in the thorax. Loss of Antp function results in altered development of the embryonic thoracic segments or can cause legs to be transformed into antennae. Certain combinations of Antp recessive lethal alleles complement to permit normal development. The structure of the Antp gene, analyzed by sequencing cDNA clones and exons and by transcript mapping, revealed some of the basis for its genetic complexity. It has two promoters governing two nested transcription units, one unit 36 and one 103 kilobase pairs (kb) long. Both units incorporated the same protein-coding exons, all of which are located in the 3'-most 13 kb of the gene. The two promoters resulted in the attachment of either of two long noncoding leader sequences (1.5 and 1.7 kb) to a 1.1-kb open reading frame. Both transcription units used the same pair of alternative polyadenylation sites 1.4 kb apart; the choice of sites was developmentally regulated. Some of the mutations that disrupt the larger transcription unit complemented a mutation affecting the smaller one. Dominant mutations that transform antennae into legs split the gene but left the coding exons intact. The encoded protein has unusually long runs of glutamine and a homeodomain near the C terminus.

scholarly journals The Helicase-Like Domain of Plant Potexvirus Replicase Participates in Formation of RNA 5′ Cap Structure by Exhibiting RNA 5′-Triphosphatase Activity

2001 ◽  
Vol 75 (24) ◽  
pp. 12114-12120 ◽  
Author(s):  
Yi-Ija Li ◽  
Ting-Wan Shih ◽  
Yau-Heiu Hsu ◽  
Yu-Tsung Han ◽  
Yih-Leh Huang ◽  
...  
Keyword(s):  
Enzymatic Activities ◽  
Nucleoside Triphosphate ◽  
Binding Motif ◽  
Helicase Domain ◽  
Cdna Fragment ◽  
Reading Frame ◽  
Metal Affinity ◽  
C Terminus ◽  
Capping Enzyme ◽  
Viral Replicase

ABSTRACT Open reading frame 1 (ORF1) of potexviruses encodes a viral replicase comprising three functional domains: a capping enzyme at the N terminus, a putative helicase in the middle, and a polymerase at the C terminus. To verify the enzymatic activities associated with the putative helicase domain, the corresponding cDNA fragment from bamboo mosaic virus (BaMV) was cloned into vector pET32 and the protein was expressed in Escherichia coli and purified by metal affinity chromatography. An activity assay confirmed that the putative helicase domain has nucleoside triphosphatase activity. We found that it also possesses an RNA 5′-triphosphatase activity that specifically removes the γ phosphate from the 5′ end of RNA. Both enzymatic activities were abolished by the mutation of the nucleoside triphosphate-binding motif (GKS), suggesting that they have a common catalytic site. A typical m7GpppG cap structure was formed at the 5′ end of the RNA substrate when the substrate was treated sequentially with the putative helicase domain and the N-terminal capping enzyme, indicating that the putative helicase domain is truly involved in the process of cap formation by exhibiting its RNA 5′-triphosphatase activity.

scholarly journals Molecular and Functional Characterization of a Unique Sucrose Hydrolase from Xanthomonas axonopodis pv. glycines

2004 ◽  
Vol 186 (2) ◽  
pp. 411-418 ◽  
Author(s):  
Hong-Suk Kim ◽  
Hyoung-Joon Park ◽  
Sunggi Heu ◽  
Jin Jung
Keyword(s):  
Insertional Mutagenesis ◽  
Catalytic Mechanism ◽  
Genomic Library ◽  
Functional Characterization ◽  
Amino Acid Sequences ◽  
Protein Coding ◽  
Reading Frame ◽  
Xanthomonas Axonopodis ◽  
Sucrose Induction

ABSTRACT A novel sucrose hydrolase (SUH) from Xanthomonas axonopodis pv. glycines, a causative agent of bacterial pustule disease on soybeans, was studied at the functional and molecular levels. SUH was shown to act rather specifically on sucrose (Km = 2.5 mM) but not on sucrose-6-phosphate. Protein analysis of purified SUH revealed that, in this monomeric enzyme with an estimated molecular mass of 70,223 ± 12 Da, amino acid sequences determined for several segments have corresponding nucleotide sequences in XAC3490, a protein-coding gene found in the genome of X. axonopodis pv. citri. Based on this information, the SUH gene, consisting of an open reading frame of 1,935 bp, was cloned by screening a genomic library of X. axonopodis pv. glycines 8ra. Database searches and sequence comparison revealed that SUH has significant homology to some family 13 enzymes, with all of the crucial invariant residues involved in the catalytic mechanism conserved, but it shows no similarity to known invertases belonging to family 32. suh expression in X. axonopodis pv. glycines requires sucrose induction, and insertional mutagenesis resulted in an absence of sucrose-inducible sucrose hydrolase activity in crude protein extracts and a sucrose-negative phenotype. Recombinant SUH, overproduced in Escherichia coli and purified, was shown to have the same enzymatic characteristics in terms of kinetic parameters.

scholarly journals Purification and characterization of two components of epoxypropane isomerase/carboxylase from Xanthobacter Py2

1996 ◽  
Vol 319 (2) ◽  
pp. 499-506 ◽  
Author(s):  
Chan K. N. CHION CHAN KWO ◽  
David J LEAK
Keyword(s):  
Amino Acid ◽  
Reductase Activity ◽  
Sequence Similarity ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Predicted Amino Acid Sequence ◽  
Reading Frame ◽  
C Terminus ◽  
A Subunit ◽  
Xanthobacter Py2

Epoxypropane isomerase from Xanthobacter Py2 has been resolved into at least two components (A and B) by ion-exchange chromatography. Both components were required for the degradation of epoxypropane and were purified further. Component A was apparently homohexameric with a subunit Mr of about 44000, and possessed NAD+-dependent dihydrolipoamide dehydrogenase activity and lipoamide reductase activity. It was sensitive to inhibition by o-phenanthroline and the thiol-specific reagents N-ethylmaleimide (NEM) and p-chloromercuribenzoate. Component B was homodimeric with a subunit Mr of 62170 and contained 2 mol·mol-1 FAD. It had an NADPH-dependent lipoamide reductase activity which was sensitive to NEM and p-chloromercuribenzoate. The N-terminal amino acid sequences and monomer sizes of components A and B correspond to those of ORF1 and ORF3 respectively (ORF = open reading frame) of a recently published sequence of a clone which complements mutants unable to degrade epoxypropane. NADPH was found to replace the need for a low-Mr fraction in epoxypropane degradation assays containing components A and B and NAD+. The predicted amino acid sequence of component A (ORF1) has been analysed and shown to contain a potential ADP binding site near the N-terminus and a putative cofactor binding domain near the C-terminus, with sequence similarity to the biotinyl and lipoyl binding domains of biotin-dependent carboxylases and 2-oxoacid dehydrogenases respectively. A reaction mechanism for epoxypropane degradation, incorporating recent evidence for combined isomerization and carboxylation to acetoacetate, is discussed.

scholarly journals Pairs of compensatory frameshifting mutations contribute to evolution of protein-coding sequences in vertebrates and insects

2020 ◽  
Author(s):  
Dmitry Biba ◽  
Galya Klink ◽  
Georgii Bazykin
Keyword(s):  
Negative Selection ◽  
Amino Acid Sequences ◽  
Fitness Cost ◽  
Vertebrate Species ◽  
High Confidence ◽  
Protein Coding ◽  
Coding Sequences ◽  
Reading Frame ◽  
Human Genes

AbstractInsertions and deletions of lengths not divisible by 3 in protein-coding sequences cause frameshifts that usually induce premature stop codons and may carry a high fitness cost. However, this cost can be circumvented by a second compensatory indel restoring the reading frame. The role of such compensatory frameshifting mutations (CFMs) in evolution has not been studied systematically. Here, we use whole-genome alignments of protein coding genes of 100 vertebrate species, and of 122 insect species, studying the prevalence of CFMs in their divergence. After stringent filtering, we detect a total of 11 high-confidence genes carrying pairs of CFMs, including three human genes: RAB36, ARHGAP6 and NCR3LG1. CFMs tended to occur in genes under relaxed negative selection, indicating that they are typically prevented at functionally important genes. In some instances, mutations closely predating or following the CFMs restored the biochemical similarity of the frameshifted segment to the ancestral sequence, possibly reducing or negating the fitness cost of a CFM. Typically, however, the resulting sequence bore no similarity to the ancestral one, indicating that the CFMs can uncover radically novel regions of sequence space. In total, CFMs represent a potentially important and previously overlooked source of novel variation in amino acid sequences.

SAC3B is a target of CML19, the centrin 2 of Arabidopsis thaliana

2020 ◽  
Vol 477 (1) ◽  
pp. 173-189 ◽  
Author(s):  
Marco Pedretti ◽  
Carolina Conter ◽  
Paola Dominici ◽  
Alessandra Astegno
Keyword(s):  
Excision Repair ◽  
Complex Structure ◽  
Binding Motif ◽  
C Terminus ◽  
Repair Protein ◽  
Nucleotide Excision ◽  
Ef Hand ◽  
Molecular Determinants ◽  
Structure In Solution

Arabidopsis centrin 2, also known as calmodulin-like protein 19 (CML19), is a member of the EF-hand superfamily of calcium (Ca2+)-binding proteins. In addition to the notion that CML19 interacts with the nucleotide excision repair protein RAD4, CML19 was suggested to be a component of the transcription export complex 2 (TREX-2) by interacting with SAC3B. However, the molecular determinants of this interaction have remained largely unknown. Herein, we identified a CML19-binding site within the C-terminus of SAC3B and characterized the binding properties of the corresponding 26-residue peptide (SAC3Bp), which exhibits the hydrophobic triad centrin-binding motif in a reversed orientation (I8W4W1). Using a combination of spectroscopic and calorimetric experiments, we shed light on the SAC3Bp–CML19 complex structure in solution. We demonstrated that the peptide interacts not only with Ca2+-saturated CML19, but also with apo-CML19 to form a protein–peptide complex with a 1 : 1 stoichiometry. Both interactions involve hydrophobic and electrostatic contributions and include the burial of Trp residues of SAC3Bp. However, the peptide likely assumes different conformations upon binding to apo-CML19 or Ca2+-CML19. Importantly, the peptide dramatically increases the affinity for Ca2+ of CML19, especially of the C-lobe, suggesting that in vivo the protein would be Ca2+-saturated and bound to SAC3B even at resting Ca2+-levels. Our results, providing direct evidence that Arabidopsis SAC3B is a CML19 target and proposing that CML19 can bind to SAC3B through its C-lobe independent of a Ca2+ stimulus, support a functional role for these proteins in TREX-2 complex and mRNA export.

scholarly journals Molecular Evolution at the decapentaplegic Locus in Drosophila

Genetics ◽  
1997 ◽  
Vol 145 (2) ◽  
pp. 297-309 ◽  
Author(s):  
Stuart J Newfeld ◽  
Richard W Padgett ◽  
Seth D Findley ◽  
Brent G Richter ◽  
Michele Sanicola ◽  
...  
Keyword(s):  
Molecular Evolution ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Selective Constraint ◽  
Amino Acid Sequences ◽  
Regulatory Sequences ◽  
Protein Coding ◽  
Terminal Ligand ◽  
Cdna Sequences ◽  
Interspecific Comparisons

Using an elaborate set of cis-regulatory sequences, the decapentaplegic (dpp) gene displays a dynamic pattern of gene expression during development. The C-terminal portion of the DPP protein is processed to generate a secreted signaling molecule belonging to the transforming growth factor-β (TGF-β) family. This signal, the DPP ligand, is able to influence the developmental fates of responsive cells in a concentration-dependent fashion. Here we examine the sequence level organization of a significant portion of the dpp locus in Drosophila melanogaster and use interspecific comparisons with D. simulans, D. pseudoobscura and D.virilis to explore the molecular evolution of the gene. Our interspecific analysis identified significant selective constraint on both the nucleotide and amino acid sequences. As expected, interspecific comparison of protein coding sequences shows that the C-terminal ligand region is highly conserved. However, the central portion of the protein is also conserved, while the N-terminal third is quite variable. Comparison of noncoding regions reveals significant stretches of nucleotide identity in the 3′ untranslated portion of exon 3 and in the intron between exons 2 and 3. An examination of cDNA sequences representing five classes of dpp transcripts indicates that these transcripts encode the same polypeptide.

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