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Crystal structures of human and Staphylococcus aureus pyruvate carboxylase and molecular insights into the carboxyltransfer reaction

Nature Structural & Molecular Biology volume 15, pages 295–302 (2008)Cite this article

Abstract

Pyruvate carboxylase (PC) catalyzes the biotin-dependent production of oxaloacetate and has important roles in gluconeogenesis, lipogenesis, insulin secretion and other cellular processes. PC contains the biotin carboxylase (BC), carboxyltransferase (CT) and biotin-carboxyl carrier protein (BCCP) domains. We report here the crystal structures at 2.8-Å resolution of full-length PC from Staphylococcus aureus and the C-terminal region (missing only the BC domain) of human PC. A conserved tetrameric association is observed for both enzymes, and our structural and mutagenesis studies reveal a previously uncharacterized domain, the PC tetramerization (PT) domain, which is important for oligomerization. A BCCP domain is located in the active site of the CT domain, providing the first molecular insights into how biotin participates in the carboxyltransfer reaction. There are dramatic differences in domain positions in the monomer and the organization of the tetramer between these enzymes and the PC from Rhizobium etli.

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Figure 1: Structure of CT+PT+BCCP domain of human pyruvate carboxylase (PC).
Figure 2: Structure of S. aureus pyruvate carboxylase (PC).
Figure 3: Dramatic differences in the tetramer interface of human and S. aureus pyruvate carboxylase (PC) compared to that of R. etli PC.
Figure 4: Binding of the biotin-carboxyl carrier protein (BCCP) domain in the active site of the carboxyltransferase (CT) domain.
Figure 5: Binding of biotin-carboxyl carrier protein (BCCP)-biotin in a previously uncharacterized exo pocket at the interface between the pyruvate carboxylase (PC) tetramerization (PT) and carboxyltransferase (CT) domains of S. aureus PC.
Figure 6: Possible conformations of the biotin-carboxyl carrier protein (BCCP) domain during catalysis.

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Acknowledgements

We thank A. Heroux and H. Robinson for setting up the X29A beamline; R. Abramowitz and J. Schwanof for setting up the X4A beamline at the NSLS; L. Yu for help with crystallization and kinetic experiments; D. Parisotto for help with kinetic assays; W.W. Cleland for helpful discussions. This research is supported in part by a grant from the US National Institutes of Health (DK67238) to L.T.

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  1. Department of Biological Sciences, 212 Amsterdam Avenue, Columbia University, New York, 10027, New York, USA

    Song Xiang & Liang Tong

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Correspondence to Liang Tong.

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Xiang, S., Tong, L. Crystal structures of human and Staphylococcus aureus pyruvate carboxylase and molecular insights into the carboxyltransfer reaction. Nat Struct Mol Biol 15, 295–302 (2008). https://doi.org/10.1038/nsmb.1393

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