scholarly journals The amino acid sequence of the small monomeric phosphoglycerate mutase from the fission yeast Schizosaccharomyces pombe

1994 ◽  
Vol 297 (3) ◽  
pp. 603-608 ◽  
Author(s):  
J Nairn ◽  
N C Price ◽  
L A Fothergill-Gilmore ◽  
G E Walker ◽  
J E Fothergill ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Phosphoglycerate Mutase ◽  
Yeast Saccharomyces Cerevisiae ◽  
Reading Frame ◽  
C Terminus ◽  
Contact Residues

The amino acid sequence of the monomeric 2,3-bisphosphoglycerate (BPG)-dependent phosphoglycerate mutase (PGAM) from the fission yeast Schizosaccharomyces pombe has been determined. Amino acid sequencing of proteolytic fragments of the enzyme showed the S. pombe mutase to be similar in sequence to the tetrameric enzyme of baker's yeast (Saccharomyces cerevisiae). An S. pombe cDNA library was screened using a PCR fragment generated from two oligonucleotides complementary to sequences encoding the regions at the two active-site histidine residues. The 0.63 kb cDNA encoded an open reading frame of 210 amino acids. This sequence agreed completely with sequences of peptides derived from the purified protein. The amino acid sequence of S. pombe PGAM is 43% identical with that of S. cerevisiae PGAM and shows an equally high degree of identity with BPG-dependent PGAMs from other sources. However, the sequence of the S. pombe enzyme differs from other BPG-dependent enzymes in three important ways: (i) it does not contain the alanine- and lysine-rich sequence of amino acids at the C-terminus which have been proposed to constitute a flexible tail involved in catalysis; (ii) the sequence spanning residues 122-146 (S. cerevisiae PGAM numbering) is not present in the S. pombe PGAM sequence; in the S. cerevisiae PGAM crystal structure this stretch of sequence has been shown to occur as an extended loop, part of which is involved in inter-subunit interactions; (iii) the amino acid sequence in the region of a second S. cerevisiae inter-subunit contact (residues 74-78) shows radical mutations in the S. pombe enzyme.

The amino acid sequence of yeast phosphoglycerate mutase

1982 ◽  
Vol 215 (1198) ◽  
pp. 19-44 ◽  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Proteolytic Enzymes ◽  
Phosphoglycerate Mutase ◽  
Crystallographic Structure ◽  
X Ray ◽  
C Terminus ◽  
Detailed Interpretation ◽  
High Degree

The complete amino acid sequence of yeast phosphoglycerate mutase comprising 241 residues has been determined. The sequence was deduced from the two cyanogen bromide fragments, and from the peptides derived from these fragments after digestion by a number of proteolytic enzymes. Determination of this sequence now allows a detailed interpretation of the existing high-resolution X-ray crystallographic structure. A comparison of the sequence reported here with the sequences of peptides from phosphoglycerate mutases from other species, and with the sequence of erythrocyte diphosphoglycerate mutase, indicates that these enzymes have a high degree of structural homology. Autolysis of phosphoglycerate mutase by yeast extracts leads to the complete loss of mutase activity, and the formation of electrophoretically distinguishable forms (R. Sasaki, E. Sugimoto & H. Chiba, Archs Biochem. Biophys. 115, 53-61 (1966)). It is apparent from the amino acid sequence that these changes are due to the loss of an 8─12 residue peptide from the C-terminus.

scholarly journals The Schizosaccharomyces pombe homolog of Saccharomyces cerevisiae HAP2 reveals selective and stringent conservation of the small essential core protein domain.

1991 ◽  
Vol 11 (2) ◽  
pp. 611-619 ◽  
Author(s):  
J T Olesen ◽  
J D Fikes ◽  
L Guarente
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Transcriptional Activation ◽  
Core Protein ◽  
Protein Domain ◽  
Yeast Saccharomyces Cerevisiae ◽  
Acid Protein ◽  
Ccaat Box

The fission yeast Schizosaccharomyces pombe is immensely diverged from budding yeast (Saccharomyces cerevisiae) on an evolutionary time scale. We have used a fission yeast library to clone a homolog of S. cerevisiae HAP2, which along with HAP3 and HAP4 forms a transcriptional activation complex that binds to the CCAAT box. The S. pombe homolog php2 (S. pombe HAP2) was obtained by functional complementation in an S. cerevisiae hap2 mutant and retains the ability to associate with HAP3 and HAP4. We have previously demonstrated that the HAP2 subunit of the CCAAT-binding transcriptional activation complex from S. cerevisiae contains a 65-amino-acid "essential core" structure that is divisible into subunit association and DNA recognition domains. Here we show that Php2 contains a 60-amino-acid block that is 82% identical to this core. The remainder of the 334-amino-acid protein is completely without homology to HAP2. The function of php2 in S. pombe was investigated by disrupting the gene. Strikingly, like HAP2 in S. cerevisiae, the S. pombe gene is specifically involved in mitochondrial function. This contrasts to the situation in mammals, in which the homologous CCAAT-binding complex is a global transcriptional activator.

The Schizosaccharomyces pombe homolog of Saccharomyces cerevisiae HAP2 reveals selective and stringent conservation of the small essential core protein domain

1991 ◽  
Vol 11 (2) ◽  
pp. 611-619
Author(s):  
J T Olesen ◽  
J D Fikes ◽  
L Guarente
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Transcriptional Activation ◽  
Core Protein ◽  
Protein Domain ◽  
Yeast Saccharomyces Cerevisiae ◽  
Acid Protein ◽  
Ccaat Box

The fission yeast Schizosaccharomyces pombe is immensely diverged from budding yeast (Saccharomyces cerevisiae) on an evolutionary time scale. We have used a fission yeast library to clone a homolog of S. cerevisiae HAP2, which along with HAP3 and HAP4 forms a transcriptional activation complex that binds to the CCAAT box. The S. pombe homolog php2 (S. pombe HAP2) was obtained by functional complementation in an S. cerevisiae hap2 mutant and retains the ability to associate with HAP3 and HAP4. We have previously demonstrated that the HAP2 subunit of the CCAAT-binding transcriptional activation complex from S. cerevisiae contains a 65-amino-acid "essential core" structure that is divisible into subunit association and DNA recognition domains. Here we show that Php2 contains a 60-amino-acid block that is 82% identical to this core. The remainder of the 334-amino-acid protein is completely without homology to HAP2. The function of php2 in S. pombe was investigated by disrupting the gene. Strikingly, like HAP2 in S. cerevisiae, the S. pombe gene is specifically involved in mitochondrial function. This contrasts to the situation in mammals, in which the homologous CCAAT-binding complex is a global transcriptional activator.

scholarly journals Phosphoprotein with Phosphoglycerate Mutase Activity from the Archaeon Sulfolobus solfataricus

2003 ◽  
Vol 185 (7) ◽  
pp. 2112-2121 ◽  
Author(s):  
M. Ben Potters ◽  
Barbara T. Solow ◽  
Kenneth M. Bischoff ◽  
David E. Graham ◽  
Brian H. Lower ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sulfolobus Solfataricus ◽  
Sodium Dodecyl ◽  
Open Reading Frame ◽  
Phosphoglycerate Mutase ◽  
Divalent Metal Ions ◽  
Reading Frame ◽  
Sds Page

ABSTRACT When soluble extracts of the extreme acidothermophilic archaeon Sulfolobus solfataricus were incubated with [γ-32P]ATP, several proteins were radiolabeled. One of the more prominent of these, which migrated with a mass of ∼46 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was purified by column chromatography and SDS-PAGE and subjected to amino acid sequence analysis via both the Edman technique and mass spectroscopy. The best match to the partial sequence obtained was the potential polypeptide product of open reading frame sso0417, whose DNA-derived amino acid sequence displayed many features reminiscent of the 2,3-diphosphoglycerate-independent phosphoglycerate (PGA) mutases [iPGMs]. Open reading frame sso0417 was therefore cloned, and its protein product was expressed in Escherichia coli. Assays of its catalytic capabilities revealed that the protein was a moderately effective PGA mutase that also exhibited low levels of phosphohydrolase activity. PGA mutase activity was dependent upon the presence of divalent metal ions such as Co2+ or Mn2+. The recombinant protein underwent autophosphorylation when incubated with either [γ-32P]ATP or [γ-32P]GTP. The site of phosphorylation was identified as Ser59, which corresponds to the catalytically essential serine residue in bacterial and eucaryal iPGMs. The phosphoenzyme intermediate behaved in a chemically and kinetically competent manner. Incubation of the 32P-labeled phosphoenzyme with 3-PGA resulted in the disappearance of radioactive phosphate and the concomitant appearance of 32P-labeled PGA at rates comparable to those measured in steady-state assays of PGA mutase activity.

scholarly journals Autophagy-deficient Schizosaccharomyces pombe mutants undergo partial sporulation during nitrogen starvation

Microbiology ◽  
2009 ◽  
Vol 155 (12) ◽  
pp. 3816-3826 ◽  
Author(s):  
Hiroyuki Mukaiyama ◽  
Shiro Kajiwara ◽  
Akira Hosomi ◽  
Yuko Giga-Hama ◽  
Naotaka Tanaka ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Nitrogen Source ◽  
Environmental Changes ◽  
Nitrogen Starvation ◽  
Nitrogen Sources ◽  
Vacuole Fusion ◽  
Sporulation Process

Autophagy is triggered when organisms sense radical environmental changes, including nutritional starvation. During autophagy, cytoplasmic components, including organelles, are enclosed within autophagosomes and are degraded upon lysosome–vacuole fusion. In this study, we show that processing of GFP-tagged Atg8 can serve as a marker for autophagy in the fission yeast Schizosaccharomyces pombe. Using this marker, 13 Atg homologues were also found to be required for autophagy in fission yeast. In budding yeast, autophagy-deficient mutants are known to be sterile, whereas in fission yeast we found that up to 30 % of autophagy-defective cells with amino acid auxotrophy were able to recover sporulation when an excess of required amino acids was supplied. Furthermore, we found that approximately 15 % of the autophagy-defective cells were also able to sporulate when a prototrophic strain was subjected to nitrogen starvation, which suggested that fission yeast may store sufficient intracellular nitrogen to allow partial sporulation under nitrogen-limiting conditions, although the majority of the nitrogen source is supplied by autophagy. Monitoring of the sporulation process revealed that the process was blocked non-specifically at various stages in the atg1Δ and atg12Δ mutants, possibly due to a shortage of amino acids. Taking advantage of this partial sporulation ability of fission yeast, we sought evidence for the existence of a recycling system for nitrogen sources during starvation.

Characterization of the extracellular poly(3-hydroxybutyrate) depolymerase ofComamonassp. and of its structural gene

1995 ◽  
Vol 41 (13) ◽  
pp. 160-169 ◽  
Author(s):  
Dieter Jendrossek ◽  
Martina Backhaus ◽  
Meike Andermann
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Binding Site ◽  
Catalytic Domain ◽  
Substrate Binding ◽  
Catalytic Triad ◽  
Reading Frame ◽  
Phb Depolymerase ◽  
Substrate Binding Site

The poly(3-hydroxybutyrate) (PHB) depolymerase structural gene of Comamonas sp. (phaZCsp) was cloned in Escherichia coli and identified by halo formation on PHB-containing solid medium. The nucleotide sequence of a 1719 base pair MboI fragment was determined and contained one large open reading frame (ORF1, 1542 base pairs). This open reading frame encoded the precursor of the PHB depolymerase (514 amino acids; Mr, 53 095), and the deduced amino acid sequence was in agreement with the N-terminal amino acid sequence of the purified PHB depolymerase from amino acid 26 onwards. Analysis of the deduced amino acid sequence revealed a domain structure of the protein: a signal peptide that was 25 amino acids long was followed by a catalytic domain of about 300 amino acids, a fibronectin type III (Fn3) modul sequence, and a putative PHB-specific substrate-binding site. By comparison of the primary structure with that of other polyhydroxyalkanoate (PHA) depolymerases, the catalytic domain apparently contained a catalytic triad of serine, histidine, and aspartate. In addition, a conserved region resembling the oxyanion hole of lipases was present. The catalytic domain was linked to a C-terminal putative substrate-binding site by a sequence about 90 amino acids long resembling the Fn3 modul of fibronectin and other eukaryotic extracellular matrix proteins. A threonine-rich region, which was found in four of five PHA depolymerases of Pseudomonas lemoignei, was not present in the Comamonas sp. depolymerase. The similarities with and differences from other PHA depolymerases are discussed.Key words: biodegradable polymer, poly(3-hydroxybutyrate) depolymerase, serine hydrolase, catalytic triad, Comamonas sp., fibronectin type III modul, substrate-binding site.

scholarly journals New Mg2+-Dependent Oxytetracycline Resistance Determinant Tet 34 in Vibrio Isolates from Marine Fish Intestinal Contents

2002 ◽  
Vol 46 (5) ◽  
pp. 1550-1552 ◽  
Author(s):  
Lisa Nonaka ◽  
Satoru Suzuki
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Active Site ◽  
Open Reading Frame ◽  
Purine Nucleotide ◽  
Chromosomal Dna ◽  
Reading Frame ◽  
Intestinal Contents ◽  
Seriola Quinqueradiata

ABSTRACT A new oxytetracycline (OTC) resistance (Otcr) determinant, Tet 34, was cloned from chromosomal DNA of Vibrio sp. no. 6 isolated from intestinal contents of cultured yellowtail (Seriola quinqueradiata). The transformant, containing cloned Tet 34, could grow in broth containing 25 μg of drug per ml with 10 mM MgCl2. Tet 34 encoded an open reading frame (ORF) 154 amino acids long. The amino acid sequence of the ORF was homologous to sequences of several bacterial xanthine-guanine phosphoribosyltransferases (XPRTs), which act in purine nucleotide salvage synthesis. Mg2+ binding site residues and the active site were highly conserved in XPRT and the ORF of Tet 34. The results suggest that Tet 34 encodes a new Mg2+-dependent Otcr mechanism.

Complete Covalent Structure of Human Platelet Factor 4

1979 ◽  
Vol 42 (05) ◽  
pp. 1652-1660 ◽  
Author(s):  
Francis J Morgan ◽  
Geoffrey S Begg ◽  
Colin N Chesterman
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Human Platelet ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Disulphide Bonds ◽  
Covalent Structure

SummaryThe amino acid sequence of the subunit of human platelet factor 4 has been determined. Human platelet factor 4 consists of identical subunits containing 70 amino acids, each with a molecular weight of 7,756. The molecule contains no methionine, phenylalanine or tryptophan. The proposed amino acid sequence of PF4 is: Glu-Ala-Glu-Glu-Asp-Gly-Asp-Leu-Gln-Cys-Leu-Cys-Val-Lys-Thr-Thr-Ser- Gln-Val-Arg-Pro-Arg-His-Ile-Thr-Ser-Leu-Glu-Val-Ile-Lys-Ala-Gly-Pro-His-Cys-Pro-Thr-Ala-Gin- Leu-Ile-Ala-Thr-Leu-Lys-Asn-Gly-Arg-Lys-Ile-Cys-Leu-Asp-Leu-Gln-Ala-Pro-Leu-Tyr-Lys-Lys- Ile-Ile-Lys-Lys-Leu-Leu-Glu-Ser. From consideration of the homology with p-thromboglobulin, disulphide bonds between residues 10 and 36 and between residues 12 and 52 can be inferred.

scholarly journals Sequence and structure of mtr, an amino acid transport gene of Neurospora crassa

Genome ◽  
1991 ◽  
Vol 34 (4) ◽  
pp. 644-651 ◽  
Author(s):  
Kenneth Koo ◽  
W. Dorsey Stuart
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Neurospora Crassa ◽  
Rna Binding ◽  
Signal Sequence ◽  
Average Length ◽  
Open Reading Frame ◽  
Mrna Transcript ◽  
S1 Nuclease ◽  
Reading Frame

The gene product of the mtr locus of Neurospora crassa is required for the transport of neutral aliphatic and aromatic amino acids via the N system. We have previously cloned three cosmids containing Neurospora DNA that complement the mtr-6(r) mutant allele. The cloned DNAs were tightly linked to restriction fragment length polymorphisms that flank the mtr locus. A 2.9-kbp fragment from one cosmid was subcloned and found to complement the mtr-6(r) allele. Here we report the sequence of the fragment that hybridized to a poly(A)+ mRNA transcript of about 2300 nucleotides. We have identified an 845-bp open reading frame (ORF) having a 59-bp intron as the potential mtr ORF. S1 nuclease analysis of the transcript confirmed the transcript size and the presence of the intron. A second open reading frame was found upstream in the same reading frame as the mtr ORF and appears to be present in the mRNA transcript. The mtr ORF is predicted to encode a 261 amino acid polypeptide with a molecular mass of 28 613 Da. The proposed polypeptide exhibits six potential α-helical transmembrane domains with an average length of 23 amino acids, does not have a signal sequence, and contains amino acid sequence homologous to an RNA binding motif.Key words: sequence, membranes, ribonucleoprotein.

scholarly journals Terminal deoxynucleotidyltransferase. Alignment of α- and β-subunits of the core enzyme along the primary translation product

1985 ◽  
Vol 227 (3) ◽  
pp. 1003-1007 ◽  
Author(s):  
C M Beach ◽  
S K Chan ◽  
T C Vanaman ◽  
M S Coleman
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Beta Subunits ◽  
Isolation And Purification ◽  
C Terminus ◽  
Human Lymphoblast ◽  
Catalytically Active ◽  
Single Polypeptide ◽  
Dna Nucleotide Sequence

Terminal deoxynucleotidyltransferase exists in multiple Mr forms, all apparently generated from a single polypeptide of 62kDa. On isolation and purification, the smallest catalytically active protein of this enzyme consists of two subunits, alpha (12kDa) and beta (30kDa). Recently a complementary-DNA nucleotide sequence has been reported for a portion of the enzyme from human lymphoblast. We have pinpointed the locations of the alpha- and beta-subunits within the elucidated nucleotide sequence. From these data, the portions of the nucleotide sequence coding for the catalytically important area of the transferase can be estimated. Here the amino acid sequence of a number of tryptic peptides from calf alpha- and beta-subunits is presented. Because of the striking homology between the amino acid sequence of the calf enzyme and that predicted for human lymphoblast enzyme, it is possible for us to conclude that the alpha-subunit was generated from the C-terminus of the precursor protein and the beta-subunit was non-overlapping and proximal.

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