Crystal structure of the α6β6 holoenzyme of propionyl-coenzyme A carboxylase


Propionyl-coenzyme A carboxylase (PCC), a mitochondrial biotin-dependent enzyme, is essential for the catabolism of the amino acids Thr, Val, Ile and Met, cholesterol and fatty acids with an odd number of carbon atoms. Deficiencies in PCC activity in humans are linked to the disease propionic acidaemia, an autosomal recessive disorder that can be fatal in infants1,2,3,4. The holoenzyme of PCC is an α6β6 dodecamer, with a molecular mass of 750 kDa. The α-subunit contains the biotin carboxylase (BC) and biotin carboxyl carrier protein (BCCP) domains, whereas the β-subunit supplies the carboxyltransferase (CT) activity. Here we report the crystal structure at 3.2-Å resolution of a bacterial PCC α6β6 holoenzyme as well as cryo-electron microscopy (cryo-EM) reconstruction at 15-Å resolution demonstrating a similar structure for human PCC. The structure defines the overall architecture of PCC and reveals unexpectedly that the α-subunits are arranged as monomers in the holoenzyme, decorating a central β6 hexamer. A hitherto unrecognized domain in the α-subunit, formed by residues between the BC and BCCP domains, is crucial for interactions with the β-subunit. We have named it the BT domain. The structure reveals for the first time the relative positions of the BC and CT active sites in the holoenzyme. They are separated by approximately 55 Å, indicating that the entire BCCP domain must translocate during catalysis. The BCCP domain is located in the active site of the β-subunit in the current structure, providing insight for its involvement in the CT reaction. The structural information establishes a molecular basis for understanding the large collection of disease-causing mutations in PCC and is relevant for the holoenzymes of other biotin-dependent carboxylases, including 3-methylcrotonyl-CoA carboxylase (MCC)5,6,7 and eukaryotic acetyl-CoA carboxylase (ACC)8,9.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Structure of the PCC holoenzyme.
Figure 2: Interactions between the α- and β-subunits in the PCC holoenzyme.
Figure 3: The active sites of the PCC holoenzyme.
Figure 4: Locations of disease-causing mutations in the PCC holoenzyme.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates are deposited in Protein Data Bank under accession number 3N6R.


  1. 1

    Desviat, L. R. et al. Propionic acidemia: mutation update and functional and structural effects of the variant alleles. Mol. Genet. Metab. 83, 28–37 (2004)

    CAS  Article  Google Scholar 

  2. 2

    Rodriguez-Pombo, P. et al. Towards a model to explain the intragenic complementation in the heteromultimeric protein propionyl-CoA carboxylase. Biochim. Biophys. Acta 1740, 489–498 (2005)

    CAS  Article  Google Scholar 

  3. 3

    Deodato, F., Boenzi, S., Santorelli, F. M. & Dionisi-Vici, C. Methylmalonic and propionic aciduria. Am. J. Med. Genet. C. Semin. Med. Genet. 142, 104–112 (2006)

    Article  Google Scholar 

  4. 4

    Desviat, L. R. et al. New splicing mutations in propionic acidemia. J. Hum. Genet. 51, 992–997 (2006)

    CAS  Article  Google Scholar 

  5. 5

    Desviat, L. R. et al. Functional analysis of MCCA and MCCB mutations causing methylcrotonylglycinuria. Mol. Genet. Metab. 80, 315–320 (2003)

    CAS  Article  Google Scholar 

  6. 6

    Stadler, S. C. et al. Newborn screening for 3-methylcrotonyl-CoA carboxylase deficiency: population heterogeneity of MCCA and MCCB mutations and impact on risk assessment. Hum. Mutat. 27, 748–759 (2006)

    CAS  Article  Google Scholar 

  7. 7

    Stucki, M., Suormala, T., Fowler, B., Valle, D. & Baumgartner, M. R. Cryptic exon activation by disruption of exon splice enhancer. Novel mechanism causing 3-methylcrotonyl-CoA carboxylase deficiency. J. Biol. Chem. 284, 28953–28957 (2009)

    CAS  Article  Google Scholar 

  8. 8

    Wakil, S. J., Stoops, J. K. & Joshi, V. C. Fatty acid synthesis and its regulation. Annu. Rev. Biochem. 52, 537–579 (1983)

    CAS  Article  Google Scholar 

  9. 9

    Tong, L. Acetyl-coenzyme A carboxylase: crucial metabolic enzyme and attractive target for drug discovery. Cell. Mol. Life Sci. 62, 1784–1803 (2005)

    CAS  Article  Google Scholar 

  10. 10

    Waldrop, G. L., Rayment, I. & Holden, H. M. Three-dimensional structure of the biotin carboxylase subunit of acetyl-CoA carboxylase. Biochem. 33, 10249–10256 (1994)

    CAS  Article  Google Scholar 

  11. 11

    Cronan, J. E., Jr & Waldrop, G. L. Multi-subunit acetyl-CoA carboxylases. Prog. Lipid Res. 41, 407–435 (2002)

    CAS  Article  Google Scholar 

  12. 12

    Zhang, H., Yang, Z., Shen, Y. & Tong, L. Crystal structure of the carboxyltransferase domain of acetyl-coenzyme A carboxylase. Science 299, 2064–2067 (2003)

    CAS  ADS  Article  Google Scholar 

  13. 13

    Holm, L., Kaariainen, S., Rosenstrom, P. & Schenkel, A. Searching protein structure databases with DaliLite v.3. Bioinformatics 24, 2780–2781 (2008)

    CAS  Article  Google Scholar 

  14. 14

    Xiang, S. & Tong, L. Crystal structures of human and Staphylococcus aureus pyruvate carboxylase and molecular insights into the carboxyltransfer reaction. Nature Struct. Mol. Biol. 15, 295–302 (2008)

    CAS  Article  Google Scholar 

  15. 15

    Janiyani, K., Bordelon, T., Waldrop, G. L. & Cronan, J. E., Jr Function of Escherichia coli biotin carboxylase requires catalytic activity of both subunits of the homodimer. J. Biol. Chem. 276, 29864–29870 (2001)

    CAS  Article  Google Scholar 

  16. 16

    Shen, Y., Chou, C.-Y., Chang, G.-G. & Tong, L. Is dimerization required for the catalytic activity of bacterial biotin carboxylase? Mol. Cell 22, 807–818 (2006)

    CAS  Article  Google Scholar 

  17. 17

    St. Maurice, M. et al. Domain architecture of pyruvate carboxylase, a biotin-dependent multifunctional enzyme. Science 317, 1076–1079 (2007)

    CAS  ADS  Article  Google Scholar 

  18. 18

    Chou, C.-Y., Yu, L. P. C. & Tong, L. Crystal structure of biotin carboxylase in complex with substrates and implications for its catalytic mechanism. J. Biol. Chem. 284, 11690–11697 (2009)

    CAS  Article  Google Scholar 

  19. 19

    Diacovich, L. et al. Crystal structure of the b-subunit of acyl-CoA carboxylase: structure-based engineering of substrate specificity. Biochem. 43, 14027–14036 (2004)

    CAS  Article  Google Scholar 

  20. 20

    Lin, T. W. et al. Structure-based inhibitor design of AccD5, an essential acyl-CoA carboxylase carboxyltransferase domain of Mycobacterium tuberculosis. Proc. Natl Acad. Sci. USA 103, 3072–3077 (2006)

    CAS  ADS  Article  Google Scholar 

  21. 21

    Hall, P. R. et al. Transcarboxylase 12S crystal structure: hexamer assembly and substrate binding to a multienzyme core. EMBO J. 22, 2334–2347 (2003)

    CAS  Article  Google Scholar 

  22. 22

    Bilder, P. et al. The structure of the carboxyltransferase component of acetyl-CoA carboxylase reveals a zinc-binding motif unique to the bacterial enzyme. Biochem. 45, 1712–1722 (2006)

    CAS  Article  Google Scholar 

  23. 23

    Wendt, K. S., Schall, I., Huber, R., Buckel, W. & Jacob, U. Crystal structure of the carboxyltransferase subunit of the bacterial sodium ion pump glutaconyl-coenzyme A decarboxylase. EMBO J. 22, 3493–3502 (2003)

    CAS  Article  Google Scholar 

  24. 24

    Sloane, V. & Waldrop, G. L. Kinetic characterization of mutations found in propionic acidemia and methylcrotonylglycinuria. J. Biol. Chem. 279, 15772–15778 (2004)

    CAS  Article  Google Scholar 

  25. 25

    Jiang, H., Rao, K. S., Yee, V. C. & Kraus, J. P. Characterization of four variant forms of human propionyl-CoA carboxylase expressed in Escherichia coli. J. Biol. Chem. 280, 27719–27727 (2005)

    CAS  Article  Google Scholar 

  26. 26

    Muro, S. et al. Effect of PCCB gene mutations on the heteromeric and homomeric assembly of propionyl-CoA carboxylase. Mol. Genet. Metab. 74, 476–483 (2001)

    CAS  Article  Google Scholar 

  27. 27

    Perez-Cerda, C. et al. Functional analysis of PCCB mutations causing propionic acidemia based on expression studies in deficient human skin fibroblasts. Biochim. Biophys. Acta 1638, 43–49 (2003)

    CAS  Article  Google Scholar 

  28. 28

    Shen, Y., Volrath, S. L., Weatherly, S. C., Elich, T. D. & Tong, L. A mechanism for the potent inhibition of eukaryotic acetyl-coenzyme A carboxylase by soraphen A, a macrocyclic polyketide natural product. Mol. Cell 16, 881–891 (2004)

    CAS  Article  Google Scholar 

  29. 29

    Weatherly, S. C., Volrath, S. L. & Elich, T. D. Expression and characterization of recombinant fungal acetyl-CoA carboxylase and isolation of a soraphen-binding domain. Biochem. J. 380, 105–110 (2004)

    CAS  Article  Google Scholar 

  30. 30

    Pettersen, E. F. et al. UCSF Chimera – a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

    CAS  Article  Google Scholar 

  31. 31

    Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    CAS  Article  Google Scholar 

  32. 32

    McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007)

    CAS  Article  Google Scholar 

  33. 33

    Thoden, J. B., Blanchard, C. Z., Holden, H. M. & Waldrop, G. L. Movement of the biotin carboxylase B-domain as a result of ATP binding. J. Biol. Chem. 275, 16183–16190 (2000)

    CAS  Article  Google Scholar 

  34. 34

    Diacovich, L. et al. Crystal structure of the β-subunit of acyl-CoA carboxylase: structure-based engineering of substrate specificity. Biochemistry 43, 14027–14036 (2004)

    CAS  Article  Google Scholar 

  35. 35

    Xiang, S. & Tong, L. Crystal structures of human and Staphylococcus aureus pyruvate carboxylase and molecular insights into the carboxyltransfer reaction. Nature Struct. Mol. Biol. 15, 295–302 (2008)

    CAS  Article  Google Scholar 

  36. 36

    CCP4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

  37. 37

    Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

    Article  Google Scholar 

  38. 38

    Brunger, A. T. et al. Crystallography & NMR System: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    CAS  Article  Google Scholar 

  39. 39

    Jogl, G., Tao, X., Xu, Y. & Tong, L. COMO: a program for combined molecular replacement. Acta Crystallogr. D 57, 1127–1134 (2001)

    CAS  Article  Google Scholar 

  40. 40

    Ludtke, S. J., Baldwin, P. R. & Chiu, W. EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999)

    CAS  Article  Google Scholar 

  41. 41

    Pettersen, E. F. et al. UCSF Chimera – a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

    CAS  Article  Google Scholar 

  42. 42

    Blanchard, C. Z., Lee, Y. M., Frantom, P. A. & Waldrop, G. L. Mutations at four active site residues of biotin carboxylase abolish substrate-induced synergism by biotin. Biochemistry 38, 3393–3400 (1999)

    CAS  Article  Google Scholar 

Download references


We thank N. Whalen and H. Robinson for access to the X29A beamline at the National Synchrotron Light Source; J. Schwanof and R. Abramowitz for access to the X4A beamline; M. Sampat for help during the initial stages of the project; and W.W. Cleland for discussions. This research was supported in part by National Institutes of Health grants DK067238 (to L.T.), GM071940 and AI069015 (to Z.H.Z.). C.S.H. was also supported by a National Institutes of Health training program in molecular biophysics (GM08281).

Author information




C.S.H., K.S.-B., Y.S. and B.D. performed the experiments, analysed the data and commented on the manuscript. L.T. and Z.H.Z. designed and performed the experiments, analysed the data and wrote the manuscript.

Corresponding author

Correspondence to Liang Tong.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Text, Supplementary Tables 1-4, References and Supplementary Figures 1-17 with legends. (PDF 7349 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Huang, C., Sadre-Bazzaz, K., Shen, Y. et al. Crystal structure of the α6β6 holoenzyme of propionyl-coenzyme A carboxylase. Nature 466, 1001–1005 (2010).

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.