Difference between revisions of "Sandbox"

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−
* '''Description:''' phosphoglycerate mutase, glycolytic/ gluconeogenic enzyme<br/><br/>
+
* '''Description:''' pyruvate kinase, glycolytic enzyme <br/><br/>
  
 
{| align="right" border="1" cellpadding="2"  
 
{| align="right" border="1" cellpadding="2"  
 
|-
 
|-
 
|style="background:#ABCDEF;" align="center"|'''Gene name'''
 
|style="background:#ABCDEF;" align="center"|'''Gene name'''
−
|''pgm''
+
|''pyk''
 
|-
 
|-
−
|style="background:#ABCDEF;" align="center"| '''Synonyms''' || ''gpmI''
+
|style="background:#ABCDEF;" align="center"| '''Synonyms''' || ''pykA''
 
|-
 
|-
−
|style="background:#ABCDEF;" align="center"| '''Essential''' || yes
+
|style="background:#ABCDEF;" align="center"| '''Essential''' || no
 
|-
 
|-
−
|style="background:#ABCDEF;" align="center"| '''Product''' || 2,3-bisphosphoglycerate-independent phosphoglycerate mutase
+
|style="background:#ABCDEF;" align="center"| '''Product''' || pyruvate kinase
 
|-
 
|-
−
|style="background:#ABCDEF;" align="center"|'''Function''' || enzyme in glycolysis/ gluconeogenesis
+
|style="background:#ABCDEF;" align="center"|'''Function''' || catabolic enzyme in glycolysis
 
|-
 
|-
−
|style="background:#ABCDEF;" align="center"| '''MW, pI''' || 56,1 kDa, 5.21
+
|style="background:#ABCDEF;" align="center"| '''MW, pI''' || 61,9 kDa, 4.88
 
|-
 
|-
−
|style="background:#ABCDEF;" align="center"| '''Gene length, protein length''' || 1533 bp, 511 amino acids
+
|style="background:#ABCDEF;" align="center"| '''Gene length, protein length''' || 1755 bp, 585 amino acids
 
|-
 
|-
−
|style="background:#ABCDEF;" align="center"|'''Immediate neighbours''' || ''[[tpi]]'', ''[[eno]]''
+
|style="background:#ABCDEF;" align="center"|'''Immediate neighbours''' || ''[[pfkA]]'', ''[[ytzA]]''
 
|-
 
|-
−
|style="background:#FAF8CC;" align="center"|'''[http://subtiwiki.uni-goettingen.de/pgm_nucleotide.txt    Gene sequence      (+200bp)  ]'''  
+
|style="background:#FAF8CC;" align="center"|'''[http://subtiwiki.uni-goettingen.de/pyk_nucleotide.txt    Gene sequence      (+200bp)  ]'''  
−
|style="background:#FAF8CC;" align="center"|'''[http://subtiwiki.uni-goettingen.de/pgm_protein.txt Protein sequence]'''
+
|style="background:#FAF8CC;" align="center"|'''[http://subtiwiki.uni-goettingen.de/pyk_protein.txt Protein sequence]'''
 
|-
 
|-
−
|colspan="2" | '''Genetic context''' <br/> [[Image:pgm_context.gif]]
+
|colspan="2" | '''Genetic context''' <br/> [[Image:pyk_context.gif]]
 
  <div align="right"> <small>This image was kindly provided by [http://genolist.pasteur.fr/SubtiList/ SubtiList]</small></div>
 
  <div align="right"> <small>This image was kindly provided by [http://genolist.pasteur.fr/SubtiList/ SubtiList]</small></div>
 
|-
 
|-
Line 31: Line 31:
  
 
<br/><br/>
 
<br/><br/>
−
 
  
 
=The gene=
 
=The gene=
Line 37: Line 36:
 
=== Basic information ===
 
=== Basic information ===
  
−
* '''Coordinates:''' 3476911 - 3478443
+
* '''Coordinates:''' 2983830 - 2985584
  
 
===Phenotypes of a mutant ===
 
===Phenotypes of a mutant ===
  
−
essential [http://www.ncbi.nlm.nih.gov/pubmed/12682299 PubMed]
+
Unable to grow with non-PTS carbohydrates (such as glucitol or glycerol) as single carbon source.
  
 
=== Database entries ===
 
=== Database entries ===
  
−
* '''DBTBS entry:''' [http://dbtbs.hgc.jp/COG/prom/cggR-gapA-pgk-tpiA-pgm-eno.html]
+
* '''DBTBS entry:''' [http://dbtbs.hgc.jp/COG/prom/pfkA-pyk-ytzA.html]
  
−
* '''SubtiList entry:''' [http://genolist.pasteur.fr/SubtiList/genome.cgi?gene_detail+BG10898]
+
* '''SubtiList entry:''' [http://genolist.pasteur.fr/SubtiList/genome.cgi?gene_detail+BG12661]
  
 
=== Additional information===
 
=== Additional information===
Line 55: Line 54:
 
=== Basic information/ Evolution ===
 
=== Basic information/ Evolution ===
  
−
* '''Catalyzed reaction/ biological activity:''' 2-phospho-D-glycerate = 3-phospho-D-glycerate
+
* '''Catalyzed reaction/ biological activity:'''ADP + phosphoenolpyruvate --> ATP + pyruvate, the reaction is irreversible under physiological conditions
  
−
* '''Protein family:''' BPG-independent phosphoglycerate mutase family
+
* '''Protein family:''' pyruvate kinase family, (C-terminal section: PEP-utilizing enzyme family)
  
 
* '''Paralogous protein(s):'''
 
* '''Paralogous protein(s):'''
Line 67: Line 66:
 
* '''Domains:'''  
 
* '''Domains:'''  
  
−
* '''Modification:''' phosphorylation on Ser-62 [http://www.ncbi.nlm.nih.gov/sites/entrez/17218307 PubMed], [http://www.ncbi.nlm.nih.gov/pubmed/17726680 PubMed]
+
* '''Modification:''' phosphorylation (Ser36 and Ser535 or 546) [http://www.ncbi.nlm.nih.gov/pubmed/17218307 PubMed], [http://www.ncbi.nlm.nih.gov/pubmed/16493705 PubMed], [http://www.ncbi.nlm.nih.gov/pubmed/16493705 PubMed], [http://www.ncbi.nlm.nih.gov/pubmed/17726680 PubMed]
  
−
* '''Cofactor(s):''' 2 manganese ions per subunit
+
* '''Cofactor(s):''' magnesium ion, potassium
  
−
* '''Effectors of protein activity:''' inhibited by heavy-metal ions, 2,3-butanedione and sulfhydryl agents [http://www.ncbi.nlm.nih.gov/sites/entrez/33963 PubMed]
+
* '''Effectors of protein activity:''' activated by PEP [http://www.ncbi.nlm.nih.gov/sites/entrez/4623707 PubMed]
  
−
* '''Interactions:''' Pgm-[[PfkA]]
+
* '''Interactions:'''  
  
−
* '''Localization:''' Cytoplasm (Homogeneous) [http://www.ncbi.nlm.nih.gov/sites/entrez/16479537 PubMed]
+
* '''Localization:''' cytoplasm [http://www.ncbi.nlm.nih.gov/sites/entrez/16479537 PubMed]
  
 
=== Database entries ===
 
=== Database entries ===
  
−
* '''Structure:''' ''Geobacillus stearothermophilus'', complex with 2-phosphoglycerate [http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?Dopt=s&uid=16359 NCBI], ''Geobacillus stearothermophilus'', complex with 3-phosphoglycerate [http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?Dopt=s&uid=15578 NCBI]
+
* '''Structure:''' 2E28 (from ''Geobacillus stearothermophilus'') [http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?Dopt=s&uid=62183 NCBI]
  
−
* '''Swiss prot entry:''' [http://www.expasy.ch/cgi-bin/sprot-search-ac?P39773]
+
* '''Swiss prot entry:''' [http://www.uniprot.org/uniprot/P80885]
  
−
* '''KEGG entry:''' [http://www.genome.jp/dbget-bin/www_bget?bsu+BSU33910]
+
* '''KEGG entry:''' [http://www.genome.jp/dbget-bin/www_bget?bsu+BSU29180]
  
−
* '''E.C. number:''' [http://www.expasy.org/enzyme/5.4.2.1]
+
* '''E.C. number:''' [http://www.expasy.org/enzyme/2.7.1.40]
  
 
=== Additional information===
 
=== Additional information===
−
is pH sensitive
+
 
  
 
=Expression and regulation=
 
=Expression and regulation=
  
−
* '''Operon:'''  
+
* '''Operon:''' ''[[pfkA]] [[pyk]]'' [http://www.ncbi.nlm.nih.gov/sites/entrez/11489127 PubMed]
−
** ''[[cggR]]-[[gapA]]-[[pgk]]-[[tpiA]]-[[pgm]]-[[eno]]''
 
−
** ''[[pgk]]-[[tpiA]]-[[pgm]]-[[eno]]''
 
−
 
 
−
* '''Sigma factor:''' [[SigA]]
 
  
−
* '''Regulation:'''  
+
* '''Sigma factor:'''  
−
''[[cggR]]'': neg. regulated by [[CggR]] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=+11489127 PubMed], induced by sugar
 
  
−
''[[pgk]]'': constitutive [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=+11489127 PubMed]
+
* '''Regulation:''' twofold induced by glucose [http://www.ncbi.nlm.nih.gov/sites/entrez/11489127 PubMed]
  
−
* '''Regulatory mechanism:''' transcription repression by [[CggR]] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=+11489127 PubMed]
+
* '''Regulatory mechanism:'''  
  
 
* '''Additional information:'''
 
* '''Additional information:'''
Line 109: Line 103:
 
=Biological materials =
 
=Biological materials =
  
−
* '''Mutant:'''
+
* '''Mutant:''' GP590 (cat), available in [[Stülke]] lab
 +
 
 +
* '''Expression vector:'''
 +
Expression in ''E. coli'', N-terminal His-tag: pGP1100 (in [[pWH844]]), available in [[Stülke]] lab
 +
 
 +
Expression in ''B. subtilis'', native protein: pGP1411 (in [[pBQ200]]), available in [[Stülke]] lab
 +
 
 +
Expression in ''B. subtilis'', N-terminal Strep-tag: pGP1409 (in [[pGP380]]), available in [[Stülke]] lab
 +
 
 +
Expression in ''B. subtilis'', C-terminal Strep-tag: pGP1410 (in [[pGP382]]), available in [[Stülke]] lab
  
−
* '''Expression vector:''' pGP1101 (N-terminal His-tag, in [[pWH844]]), pGP396 (Pgm-S62A, N-terminal His-tag, in [[pWH844]]), pGP92 (N-terminal Strep-tag, for SPINE, expression in B. subtilis, in [[pGP380]]), available in [[Stülke]] lab
+
* '''lacZ fusion:''' see ''[[pfkA]]''
−
 
−
* '''lacZ fusion:'''
 
  
 
* '''GFP fusion:'''
 
* '''GFP fusion:'''
Line 124: Line 125:
  
 
[[Stülke|Jörg Stülke]], University of Göttingen, Germany [http://wwwuser.gwdg.de/~genmibio/stuelke.html Homepage]
 
[[Stülke|Jörg Stülke]], University of Göttingen, Germany [http://wwwuser.gwdg.de/~genmibio/stuelke.html Homepage]
−
 
−
[[Jedrzejas|Mark J. Jedrzejas]], Research Center Oakland, CA, USA  [http://www.chori.org/Principal_Investigators/Jedrzejas_Mark_J/jedrzejas_research.html Homepage]
 
  
 
=Your additional remarks=
 
=Your additional remarks=
Line 132: Line 131:
  
 
# Eymann et al. (2007) Dynamics of protein phosphorylation on Ser/Thr/Tyr in ''Bacillus subtilis''. ''Proteomics'' '''7:''' 3509-3526. [http://www.ncbi.nlm.nih.gov/pubmed/17726680 PubMed]
 
# Eymann et al. (2007) Dynamics of protein phosphorylation on Ser/Thr/Tyr in ''Bacillus subtilis''. ''Proteomics'' '''7:''' 3509-3526. [http://www.ncbi.nlm.nih.gov/pubmed/17726680 PubMed]
−
# Meile et al. (2006) Systematic localisation of proteins fused to the green fluorescent protein in ''Bacillus subtilis'': identification of new proteins at the DNA replication factory ''Proteomics'' '''6:''' 2135-2146. [http://www.ncbi.nlm.nih.gov/sites/entrez/16479537 PubMed]
+
# L&#233;vine et al. (2006) Analysis of the dynamic ''Bacillus subtilis'' Ser/Thr/Tyr phosphoproteome implicated in a wide variety of cellular processes. ''Proteomics'' '''6:''' 2157-2173 [http://www.ncbi.nlm.nih.gov/pubmed/16493705 PubMed]
−
# Jannière, L., Canceill, D., Suski, C., Kanga, S., Dalmais, B., Lestini, R., Monnier, A. F., Chapuis, J., Bolotin, A., Titok, M., Le Chatelier, E., and Ehrlich, S. D. (2007) Genetic evidence for a link between glycolysis and DNA replication. PLoS ONE 2, e447. [http://www.ncbi.nlm.nih.gov/sites/entrez/17505547 PubMed]
+
# L&#233;vine et al. (2006) Analysis of the dynamic ''Bacillus subtilis'' Ser/Thr/Tyr phosphoproteome implicated in a wide variety of cellular processes. ''Proteomics'' '''6:''' 2157-2173 [http://www.ncbi.nlm.nih.gov/pubmed/16493705 PubMed]
−
# Leyva-Vazquez, M. A., and Setlow, P. (1994) Cloning and nucleotide sequences of the genes encoding triose phosphate isomerase, phosphoglycerate mutase, and enolase from Bacillus subtilis. J Bacteriol 176: 3903-3910. [http://www.ncbi.nlm.nih.gov/sites/entrez/8021172  PubMed]
+
# Ludwig, H., Homuth, G., Schmalisch, M., Dyka, F. M., Hecker, M., and Stülke, J. (2001) Transcription of glycolytic genes and operons in ''Bacillus subtilis'': evidence for the presence of multiple levels of control of the ''gapA'' operon. Mol Microbiol 41, 409-422.[http://www.ncbi.nlm.nih.gov/sites/entrez/11489127 PubMed]
−
# Ludwig, H., Homuth, G., Schmalisch, M., Dyka, F. M., Hecker, M. & Stülke, J. (2001) Transcription of glycolytic genes and operons in Bacillus subtilis: Evidence for the presence of multiple levels of control of the gapA operon. Mol. Microbiol. 41: 409-422. [http://www.ncbi.nlm.nih.gov/sites/entrez/11489127 PubMed]
+
# Jannière, L., Canceill, D., Suski, C., Kanga, S., Dalmais, B., Lestini, R., Monnier, A. F., Chapuis, J., Bolotin, A., Titok, M., Le Chatelier, E., and Ehrlich, S. D. (2007) Genetic evidence for a link between glycolysis and DNA replication. PLoS ONE 2, e447. [http://www.ncbi.nlm.nih.gov/sites/entrez/17505547 PubMed]  
−
# Chandler et al. (1999) Structural studies on a 2,3-diphosphoglycerate independent phosphoglycerate mutase from Bacillus stearothermophilus. J. Struct. Biol. 126: 156-165. [http://www.ncbi.nlm.nih.gov/sites/entrez/10388626 PubMed]
+
# Fry, B., Zhu, T., Domach, M. M., Koepsel, R. R., Phalakornkule, C., and Ataai, M. M. (2000) Characterization of growth and acid formation in a Bacillus subtilis pyruvate kinase mutant. Appl Env Microbiol 66: 4045-4049. [http://www.ncbi.nlm.nih.gov/sites/entrez/10966427 PubMed]
−
# Jedrzejas et al. (2000) Structure and mechanism of action of a novel phosphoglycerate mutase from Bacillus stearothermophilus. EMBO J. 19: 1419-1431. [http://www.ncbi.nlm.nih.gov/sites/entrez/10747010 PubMed]
 
−
# Jedrzejas et al. (2000) Mechanism of catalysis of the cofactor-independent phosphoglycerate mutase from Bacillus stearothermophilus. Crystal structure of the complex with a 2-phosphoglycerate. J. Biol. Chem. 275: 23146-23153. [http://www.ncbi.nlm.nih.gov/sites/entrez/10764795 PubMed]
 
−
# Jedrzejas and Setlow (2001)  Comparison of the binuclear metalloenzymes diphosphoglycerate-independent phosphoglycerate mutase and alkaline phosphatase: their mechanism of catalysis via a phosphoserine intermediate. Chem. Rev. 101: 607-618. [http://www.ncbi.nlm.nih.gov/sites/entrez/11712498 PubMed]
 
−
# Ridgen et al. (2003) Insights into the catalytic mechanism of cofactor-independent phosphoglycerate mutase from X-ray crystallography, simulated dynamics and molecular modeling. J. Mol. Biol. 328: 909-920. [http://www.ncbi.nlm.nih.gov/sites/entrez/12729763 PubMed]
 
−
# Nukui et al. (2007) Structure and molecular mechanism of Bacillus anthracis cofactor-independent phosphoglycerate mutase: a crucial enzyme for spores and growing cells of Bacillus species. Biophys. J. 92: 977-988. [http://www.ncbi.nlm.nih.gov/sites/entrez/17085493 PubMed]
 
 
# Macek et al. (2007) The serine/ threonine/ tyrosine phosphoproteome of the model  bacterium ''Bacillus subtilis''. Mol. Cell. Proteomics 6: 697-707  [http://www.ncbi.nlm.nih.gov/pubmed/17218307 PubMed]
 
# Macek et al. (2007) The serine/ threonine/ tyrosine phosphoproteome of the model  bacterium ''Bacillus subtilis''. Mol. Cell. Proteomics 6: 697-707  [http://www.ncbi.nlm.nih.gov/pubmed/17218307 PubMed]

Revision as of 16:10, 31 March 2009

  • Description: pyruvate kinase, glycolytic enzyme

Gene name pyk
Synonyms pykA
Essential no
Product pyruvate kinase
Function catabolic enzyme in glycolysis
MW, pI 61,9 kDa, 4.88
Gene length, protein length 1755 bp, 585 amino acids
Immediate neighbours pfkA, ytzA
Gene sequence (+200bp) Protein sequence
Genetic context
Pyk context.gif
This image was kindly provided by SubtiList



The gene

Basic information

  • Coordinates: 2983830 - 2985584

Phenotypes of a mutant

Unable to grow with non-PTS carbohydrates (such as glucitol or glycerol) as single carbon source.

Database entries

  • DBTBS entry: [1]
  • SubtiList entry: [2]

Additional information

The protein

Basic information/ Evolution

  • Catalyzed reaction/ biological activity:ADP + phosphoenolpyruvate --> ATP + pyruvate, the reaction is irreversible under physiological conditions
  • Protein family: pyruvate kinase family, (C-terminal section: PEP-utilizing enzyme family)
  • Paralogous protein(s):

Extended information on the protein

  • Kinetic information:
  • Domains:
  • Cofactor(s): magnesium ion, potassium
  • Effectors of protein activity: activated by PEP PubMed
  • Interactions:
  • Localization: cytoplasm PubMed

Database entries

  • Structure: 2E28 (from Geobacillus stearothermophilus) NCBI
  • Swiss prot entry: [3]
  • KEGG entry: [4]
  • E.C. number: [5]

Additional information

Expression and regulation

  • Sigma factor:
  • Regulation: twofold induced by glucose PubMed
  • Regulatory mechanism:
  • Additional information:

Biological materials

  • Mutant: GP590 (cat), available in Stülke lab
  • Expression vector:

Expression in E. coli, N-terminal His-tag: pGP1100 (in pWH844), available in Stülke lab

Expression in B. subtilis, native protein: pGP1411 (in pBQ200), available in Stülke lab

Expression in B. subtilis, N-terminal Strep-tag: pGP1409 (in pGP380), available in Stülke lab

Expression in B. subtilis, C-terminal Strep-tag: pGP1410 (in pGP382), available in Stülke lab

  • lacZ fusion: see pfkA
  • GFP fusion:
  • two-hybrid system: B. pertussis adenylate cyclase-based bacterial two hybrid system (BACTH), available in Stülke lab
  • Antibody:

Labs working on this gene/protein

Jörg Stülke, University of Göttingen, Germany Homepage

Your additional remarks

References

  1. Eymann et al. (2007) Dynamics of protein phosphorylation on Ser/Thr/Tyr in Bacillus subtilis. Proteomics 7: 3509-3526. PubMed
  2. Lévine et al. (2006) Analysis of the dynamic Bacillus subtilis Ser/Thr/Tyr phosphoproteome implicated in a wide variety of cellular processes. Proteomics 6: 2157-2173 PubMed
  3. Lévine et al. (2006) Analysis of the dynamic Bacillus subtilis Ser/Thr/Tyr phosphoproteome implicated in a wide variety of cellular processes. Proteomics 6: 2157-2173 PubMed
  4. Ludwig, H., Homuth, G., Schmalisch, M., Dyka, F. M., Hecker, M., and Stülke, J. (2001) Transcription of glycolytic genes and operons in Bacillus subtilis: evidence for the presence of multiple levels of control of the gapA operon. Mol Microbiol 41, 409-422.PubMed
  5. Jannière, L., Canceill, D., Suski, C., Kanga, S., Dalmais, B., Lestini, R., Monnier, A. F., Chapuis, J., Bolotin, A., Titok, M., Le Chatelier, E., and Ehrlich, S. D. (2007) Genetic evidence for a link between glycolysis and DNA replication. PLoS ONE 2, e447. PubMed
  6. Fry, B., Zhu, T., Domach, M. M., Koepsel, R. R., Phalakornkule, C., and Ataai, M. M. (2000) Characterization of growth and acid formation in a Bacillus subtilis pyruvate kinase mutant. Appl Env Microbiol 66: 4045-4049. PubMed
  7. Macek et al. (2007) The serine/ threonine/ tyrosine phosphoproteome of the model bacterium Bacillus subtilis. Mol. Cell. Proteomics 6: 697-707 PubMed
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