Computational studies of intramolecular disulfide bonded catenanes as a novel stabilizing mechanism

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Computational studies of intramolecular disulfide
bonded catenanes as a novel stabilizing mechanism
in thermophilic microbes

• August 23, 2007
• Daniel Park
• Yeates lab, MBI, UCLA
• SoCalBSI

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Today

• Intracellular disulfide abundance in
  thermophiles/hyperthermophiles
• P. aerophilum citrate synthase
• Searching for catenanes
• Results

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Importance of studying thermophilic enzymes

• Industrial applications
• Engineering heat-stable biomolecules
• Utilizing those found in nature
• Taq DNA polymerase for PCR
• Insight into protein folding mechanisms
• Evolution of thermostable proteins

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Intracellular disulfide bond abundance

• Mallick et al., 2002
• PNAS 99, pp. 9679-9684

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Presence of disulfide bonds within the
intracellular proteins of P. aerophilum

• Both lanes reduced
• Presense and absence of iodoacetamide
• Large fraction of P. aerophilum proteins contain
  disulfide bonds

Boutz et al., 2007 JMB 368, pp. 1332-1344
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Citrate synthase (PaCS) from P. aerophilum
Boutz et al., 2007 JMB 368, pp. 1332-1344
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Catenane structure of PaCS
Boutz et al., 2007 JMB 368, pp. 1332-1344
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Disulfide bonds contribution to the
thermostability of PaCS
Boutz et al., 2007 JMB 368, pp. 1332-1344
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(No Transcript)
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Cysteine abundance at terminal regions
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Alignment of thermophilic citrate synthase
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Approach
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Possible catenanes by temperature
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Cysteine abundance at terminal regions
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Clusters of orthologous groups (COG) functional
classifications

• INFORMATION STORAGE AND PROCESSING
• J Translation, ribosomal structure and
  biogenesis
• A RNA processing and modification
• K Transcription
• L Replication, recombination and repair
• B Chromatin structure and dynamics
• CELLULAR PROCESSES AND SIGNALING
• D Cell cycle control, cell division,
  chromosome
• partitioning
• Y Nuclear structure
• V Defense mechanisms
• T Signal transduction mechanisms
• M Cell wall/membrane/envelope biogenesis
• N Cell motility
• Z Cytoskeleton
• W Extracellular structures
• U Intracellular trafficking, secretion, and
  vesicular
• transport

• METABOLISM
• C Energy production and conversion
• G Carbohydrate transport and metabolism
• E Amino acid transport and metabolism
• F Nucleotide transport and metabolism
• H Coenzyme transport and metabolism
• I Lipid transport and metabolism
• P Inorganic ion transport and metabolism
• Q Secondary metabolites biosynthesis,
  transport
• and catabolism
• POORLY CHARACTERIZED
• R General function prediction only
• S Function unknown

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Possible microbial catenanes by function
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Possible microbial catenanes by function
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Possible thermophilic catenanes by function
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Possible thermophilic catenanes further
classified by COGs (top 7)
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Possible catenane among peroxiredoxin homologs?

• O COG0450 Peroxiredoxin (7)
• Thermoanaerobacter tengcongensis MB4 20808569
• Methanosaeta thermophila PT 116754713
• Pyrobaculum islandicum DSM 4184 119873344
• Pyrobaculum islandicum DSM 4184 119871684
• Pyrobaculum calidifontis JCM 11548 126458809
• Pyrobaculum arsenaticum DSM 13514 145590729
• Methanocaldococcus jannaschii DSM 2661 15668917
• C COG0372 Citrate synthase (5)
• Pyrobaculum islandicum DSM 4184 119873179
• Pyrobaculum calidifontis JCM 11548 126459178
• Pyrobaculum arsenaticum DSM 13514 145592430
• Pyrobaculum aerophilum str. IM2 18312809
• Aeropyrum pernix K1 14601576

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P. islandicum DSM 4184 peroxidasealignment with
homologs
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P. islandicum peroxidase homolog
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P. islandicum peroxidase homolog
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Future directions

• MD simulations of possible catenanes
• Determine structures of most likely catenanes by
  X-ray crystallography
• Investigate correlation between psychrophilic
  proteins and disulfide bonding

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Acknowledgements

• Todd Yeates
• Neil King
• Jason Forse
• Brian OConnor

• Jamil Momand
• Sandra Sharp
• Wendie Johnston
• Nancy Warter-Perez

• SoCalBSI program
• Ronnie Cheng
• Funded by NIH, NSF, EWD, DOE