Masaharu Ishii Carboxylation Enzymes Journal Club in the Laboratory of Applied Microbiology　 No. 1084 Carboxylation Enzymes ～In search of Future Prospects through the Overview of Reaction Mechanisms～ 11 Mar 2011 Masaharu Ishii

http://www.kegg.jp/kegg/pathway/map/map00720.html

3-Hydroxypropionate cycle PNAS 106, 21317 (2009)

3-Hydroxypropionate/4-Hydroxybutyrate cycle Similar to 3-HP cycle Specific to 3-HP/4-HB cycle 2CO2 + CoA →Acetyl-CoA 4ATP + 8[H] Science 318, 1782 (2007)

Dicarboxylate/4-Hydroxybutyrate cycle Similar to rTCA cycle Similar to 3-HP/4-HB cycle 2CO2 + CoA →Acetyl-CoA 3ATP + 8[H] PNAS 105, 7851 (2008)

Reductive TCA cycle

Carboxylation Enzymes & Autotrophic Carbon Dioxide Fixation Pathways RubisCO Calvin-Benson cycle Formate dehydrogenase Acetyl-CoA pathway Carbon monooxide dehydrogenase Acetyl-CoA carboxylase 3-Hpcycle, 3-HP/4-HB cycle Propionyl-CoA Carboxylase Pyruvate:ferredoxin oxidoreductase Dicarboxylate/4-HB cycle, Reductive TCA cycle Pyruvate (PEP) carboxylase 2-Oxoglutarate: ferredoxin oxidoreductase 2-Oxoglutarate carboxylase

Carboxylation Enzymes & Cofactors RubisCO NA Acetyl-CoA carboxylase Biotin Propionyl-CoA carboxylase Pyruvate:ferredoxin oxidoreductase Reduced ferredoxin Pyruvate carboxylase PEP carboxylase 2-Oxoglutarate: ferredoxin oxidoreductase 2-Oxoglutarate carboxylase NA: not applicable

Carboxylation Enzymes & Cofactors RubisCO NA Acetyl-CoA carboxylase Biotin Propionyl-CoA carboxylase Pyruvate:ferredoxin oxidoreductase Reduced ferredoxin Pyruvate carboxylase PEP carboxylase 2-Oxoglutarate: ferredoxin oxidoreductase 2-Oxoglutarate carboxylase NA: not applicable

Pyruvate carboxylase -Example for the Reaction Mechanism- Carboxyphosphate Biotin carboxylase Carboxybiotin Carboxy-transferase http://www.wellesley.edu/Chemistry/chem227/metabolism/kreb/biotinmech.gif

General Reaction Mechanism for the Biotin-dependent Carboxylase JBC 276, 37355-37364 (2001) Products Substrates http://guweb2.gonzaga.edu/faculty/cronk/biochem/B-index.cfm?definition=biotin_dependent_carboxylases

Biotin-dependent Carboxylases Substrate Product Name Acetyl-CoA carboxylase Propionyl-CoA carboxylase Pyruvate carboxylase 2-Oxoglutarate carboxylase

One of Exceptions Methylcrotonoyl-CoA carboxylase 3-Methylcrotonyl-CoA 3-Methylglutaconyl-CoA Methylcrotonoyl-CoA carboxylase

Biotin-dependent Carboxylase -Future Directions for Application- 　To widen up substrate specificities of “Carboxytransferases” 　To produce new “Substrates” for Carboxytransferases

Open Question-1 Why “Carbamoylphosphate” could not have a role in carboxylation reactions?

Carboxylation enzymes & Cofactors RubisCO NA Acetyl-CoA carboxylase Biotin Propionyl-CoA carboxylase Pyruvate:ferredoxin oxidoreductase Reduced ferredoxin Pyruvate carboxylase PEP carboxylase 2-Oxoglutarate: ferredoxin oxidoreductase 2-Oxoglutarate carboxylase NA: not applicable

The Main Reactions Catalyzed by Rubisco J. Exp. Bot. 2008;59:1555-1568

that can receive carboxylation reaction? Open Question-2 Is RuBP the only sugar that can receive carboxylation reaction?

Carboxylation Enzymes & Cofactors RubisCO NA Acetyl-CoA carboxylase Biotin Propionyl-CoA carboxylase Pyruvate:ferredoxin oxidoreductase Reduced ferredoxin Pyruvate carboxylase PEP carboxylase 2-Oxoglutarate: ferredoxin oxidoreductase 2-Oxoglutarate carboxylase NA: not applicable

Proposed catalytic mechanism of PEPC based on the crystal structures of maize and E.coli PEPC H11 acts just like a biotin. Matsumura H et al. Protein Engineering, Design and Selection 2006;19:409-419

Carboxylation Enzymes & Cofactors RubisCO NA Acetyl-CoA carboxylase Biotin Propionyl-CoA carboxylase Pyruvate:ferredoxin oxidoreductase Reduced ferredoxin Pyruvate carboxylase PEP carboxylase 2-Oxoglutarate: ferredoxin oxidoreductase 2-Oxoglutarate carboxylase NA: not applicable

Pyruvate: ferredoxin oxidoreductase -Basic Point (Internal Electron Transfer)- Nature Structural Biology  6, 182 - 190 (1999)

Proposed reaction scheme for POR FEBS J. 277, 501 (2010)

EPR spectra analysis As purified + Dithionite + Pyruvate + Pyruvate + CoA + Fd1 + OGOR + 2-OG + CoA + Fd1 + OGOR + 2-OG + CoA + Acetyl-CoA FEBS J. 277, 501 (2010)

Proposed reaction scheme for POR FEBS J. 277, 501 (2010)

How can we improve POR(OGOR) through Crystal Structural Information? Improvement for the electron transfer between Fd and POR (OGOR) Is it possible to improve internal electron transfer (=electron transfer within enzyme)? Improvement for the affinity of POR (OGOR) toward Acetyl-CoA (Succinyl-CoA) Improvement for the efflux of the product

In summary: Molecular basis is the same! -Nucleophilic Attack-

But tactics for the application differs! Thank you for your attention.