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

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

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

Intracellular disulfide bond abundance Mallick et al., 2002 PNAS 99, pp

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

Citrate synthase (PaCS) from P. aerophilum Boutz et al., 2007 JMB 368, pp

Catenane structure of PaCS Boutz et al., 2007 JMB 368, pp

Disulfide bonds: contribution to the thermostability of PaCS Boutz et al., 2007 JMB 368, pp

Cysteine abundance at terminal regions

Alignment of thermophilic citrate synthase

Approach

Possible catenanes by temperature

Cysteine abundance at terminal regions

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 [O] Posttranslational modification, protein turnover, chaperones 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

Possible microbial catenanes by function

Possible thermophilic catenanes by function

Possible thermophilic catenanes further classified by COGs (top 7) Functional classification COG typeNo. of catenanes [O] Posttranslational modification, protein turnover, chaperones Peroxiredoxin7 [C] Energy production and conversion Citrate synthase5 [C] Energy production and conversion Anaerobic dehydrogenase, typically selenocysteine-containing 5 [?] Unclassified5 [G] Carbohydrate transport Transketolase, N-terminal subunit4 [EP] Amino acid and inorganic ion transport ABC-type dipeptide/oligopeptide/nickel transport system 4 [E] Amino acid transport 3-dehydroquinate synthetase4

Possible catenane among peroxiredoxin homologs? [O] COG0450 Peroxiredoxin (7) Thermoanaerobacter tengcongensis MB Methanosaeta thermophila PT Pyrobaculum islandicum DSM Pyrobaculum islandicum DSM Pyrobaculum calidifontis JCM Pyrobaculum arsenaticum DSM Methanocaldococcus jannaschii DSM [C] COG0372 Citrate synthase (5) Pyrobaculum islandicum DSM Pyrobaculum calidifontis JCM Pyrobaculum arsenaticum DSM Pyrobaculum aerophilum str. IM Aeropyrum pernix K

P. islandicum DSM 4184 peroxidase: alignment with homologs

P. islandicum peroxidase homolog

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

Acknowledgements Todd Yeates Neil King Jason Forse Brian O’Connor Jamil Momand Sandra Sharp Wendie Johnston Nancy Warter-Perez SoCalBSI program Ronnie Cheng Funded by NIH, NSF, EWD, DOE