1. 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

5. 3-Hydroxypropionate cycle
PNAS 106, (2009)

6. 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)

7. 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)

8. Reductive TCA cycle

9. 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

10. 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

11. 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

12. Pyruvate carboxylase -Example for the Reaction Mechanism-
Carboxyphosphate
Biotin carboxylase
Carboxybiotin
Carboxy-transferase

13. General Reaction Mechanism for the Biotin-dependent Carboxylase
JBC 276, (2001)
Products Substrates

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

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

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

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

18. 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

19. The Main Reactions Catalyzed by Rubisco
J. Exp. Bot. 2008;59:

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

22. 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

23. 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:

24. 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

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

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

27. 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)

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

29. 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

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

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