An unanticipated architecture of the 750-kDa α6β6 holoenzyme of 3-methylcrotonyl-CoA carboxylase

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Abstract

3-Methylcrotonyl-CoA carboxylase (MCC), a member of the biotin-dependent carboxylase superfamily, is essential for the metabolism of leucine, and deficient mutations in this enzyme are linked to methylcrotonylglycinuria (MCG) and other serious diseases in humans1,2,3,4,5,6,7,8. MCC has strong sequence conservation with propionyl-CoA carboxylase (PCC), and their holoenzymes are both 750-kilodalton (kDa) α6β6 dodecamers. Therefore the architecture of the MCC holoenzyme is expected to be highly similar to that of PCC9. Here we report the crystal structures of the Pseudomonas aeruginosa MCC (PaMCC) holoenzyme, alone and in complex with coenzyme A. Surprisingly, the structures show that the architecture and overall shape of PaMCC are markedly different when compared to PCC. The α-subunits show trimeric association in the PaMCC holoenzyme, whereas they have no contacts with each other in PCC. Moreover, the positions of the two domains in the β-subunit of PaMCC are swapped relative to those in PCC. This structural information establishes a foundation for understanding the disease-causing mutations of MCC and provides new insights into the catalytic mechanism and evolution of biotin-dependent carboxylases. The large structural differences between MCC and PCC also have general implications for the relationship between sequence conservation and structural similarity.

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Figure 1: The domains of MCCβ are swapped compared to PCCβ.
Figure 2: The MCC holoenzyme has a markedly different architecture compared to PCC.
Figure 3: The BT domain mediates interactions in the MCC holoenzyme.
Figure 4: Molecular basis for catalysis and disease-causing mutations in the MCC holoenzyme.

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Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates have been deposited in the Protein Data Bank under accessions 3U9R, 3U9S and 3U9T.

References

  1. 1

    Baumgartner, M. R. et al. The molecular basis of human 3-methylcrotonyl-CoA carboxylase deficiency. J. Clin. Invest. 107, 495–504 (2001)

  2. 2

    Gallardo, M. E. et al. The molecular basis of 3-methylcrotonylglycinuria, a disorder of leucine metabolism. Am. J. Hum. Genet. 68, 334–346 (2001)

  3. 3

    Holzinger, A. et al. Cloning of the human MCCA and MCCB genes and mutations therein reveal the molecular cause of 3-methylcrotonyl-CoA carboxylase deficiency. Hum. Mol. Genet. 10, 1299–1306 (2001)

  4. 4

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

  5. 5

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

  6. 6

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

  7. 7

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

  8. 8

    Jitrapakdee, S. et al. Structure, mechanism and regulation of pyruvate carboxylase. Biochem. J. 413, 369–387 (2008)

  9. 9

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

  10. 10

    Förster-Fromme, K. & Jendrossek, D. Catabolism of citronellol and related acyclic terpenoids in pseudomonads. Appl. Microbiol. Biotechnol. 87, 859–869 (2010)

  11. 11

    Aguilar, J. A. et al. Substrate specificity of the 3-methylcrotonyl coenzyme A (CoA) and geranyl-CoA carboxylases from Pseudomonas aeruginosa. J. Bacteriol. 190, 4888–4893 (2008)

  12. 12

    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)

  13. 13

    Kress, D. et al. An asymmetric model for Na+-translocating glutaconyl-CoA decarboxylase. J. Biol. Chem. 284, 28401–28409 (2009)

  14. 14

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

  15. 15

    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)

  16. 16

    Yu, L. P. C. et al. A symmetrical tetramer for S. aureus pyruvate carboxylase in complex with coenzyme A. Structure 17, 823–832 (2009)

  17. 17

    Lasso, G. et al. Cryo-EM analysis reveals new insights into the mechanism of action of pyruvate carboxylase. Structure 18, 1300–1310 (2010)

  18. 18

    Knowles, J. R. The mechanism of biotin-dependent enzymes. Annu. Rev. Biochem. 58, 195–221 (1989)

  19. 19

    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)

  20. 20

    Nguyen, K. V., Naviaux, R. K., Patra, S., Barshop, B. A. & Nyhan, W. L. Novel mutations in the human MCCA and MCCB gene causing methylcrotonylglycinuria. Mol. Genet. Metab. 102, 218–221 (2011)

  21. 21

    Uematsu, M. et al. Novel mutations in five Japanese patients with 3-methylcrotonyl-CoA carboxylase deficiency. J. Hum. Genet. 52, 1040–1043 (2007)

  22. 22

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

  23. 23

    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)

  24. 24

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

  25. 25

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

  26. 26

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

  27. 27

    Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

  28. 28

    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)

  29. 29

    Emsley, P. & Cowtan, K. D. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

  30. 30

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

  31. 31

    Chen, J. Z. & Grigorieff, N. SIGNATURE: a single-particle selection system for molecular electron microscopy. J. Struct. Biol. 157, 168–173 (2007)

  32. 32

    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)

  33. 33

    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)

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Acknowledgements

We thank Y. Shen for carrying out initial studies on MCC; N. Whalen and S. Myers for setting up the X29A beamline at the National Synchrotron Light Source. This research was supported in part by National Institutes of Health (NIH) grants DK067238 (to L.T.) and GM071940 (to Z.H.Z.). C.S.H. was also supported by an NIH training program in molecular biophysics (GM08281).

Author information

C.S.H. carried out protein expression, purification and crystallization experiments, mutagenesis and enzymatic assays. C.S.H. and L.T. carried out crystallographic data collection and processing, structure determination and refinement. P.G. and Z.H.Z. carried out electron microscopy experiments. All authors commented on the manuscript. L.T. supervised the project, analysed the data and wrote the paper.

Correspondence to Liang Tong.

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The authors declare no competing financial interests.

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