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Structure and function of a single-chain, multi-domain long-chain acyl-CoA carboxylase

Nature volume 518, pages 120–124 (2015)Cite this article

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Abstract

Biotin-dependent carboxylases are widely distributed in nature and have important functions in the metabolism of fatty acids, amino acids, carbohydrates, cholesterol and other compounds1,2,3,4,5,6. Defective mutations in several of these enzymes have been linked to serious metabolic diseases in humans, and acetyl-CoA carboxylase is a target for drug discovery in the treatment of diabetes, cancer and other diseases7,8,9. Here we report the identification and biochemical, structural and functional characterizations of a novel single-chain (120 kDa), multi-domain biotin-dependent carboxylase in bacteria. It has preference for long-chain acyl-CoA substrates, although it is also active towards short-chain and medium-chain acyl-CoAs, and we have named it long-chain acyl-CoA carboxylase. The holoenzyme is a homo-hexamer with molecular mass of 720 kDa. The 3.0 Å crystal structure of the long-chain acyl-CoA carboxylase holoenzyme from Mycobacterium avium subspecies paratuberculosis revealed an architecture that is strikingly different from those of related biotin-dependent carboxylases10,11. In addition, the domains of each monomer have no direct contact with each other. They are instead extensively swapped in the holoenzyme, such that one cycle of catalysis involves the participation of four monomers. Functional studies in Pseudomonas aeruginosa suggest that the enzyme is involved in the utilization of selected carbon and nitrogen sources.

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Figure 1: Crystal structure of LCC from M. avium subspecies paratuberculosis (MapLCC).
Figure 2: Extensive domain swapping in the structure of the MapLCC holoenzyme.
Figure 3: Interactions between the central core and the rest of the holoenzyme.
Figure 4: Catalysis and function of LCC.

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Protein Data Bank

Data deposits

Coordinates and structure factors have been deposited in Protein Data Bank under accession number 4RCN.

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Acknowledgements

We thank C. Huang for carrying out some initial studies in this project; A. Price-Whelan for discussions on P. aeruginosa physiology; R. Jackimowicz, N. Whalen and H. Robinson for access to the X29A beamline; Z. Li for EM support; P. Penczek for help with SPARX. The in-house instrument for X-ray diffraction was purchased with a National Institutes of Health (NIH) grant to L.T. (S10OD012018). This research is supported by grants from the NIH (R01DK067238 to L.T. and R01AI103369 to L.E.P.D.) and from the Protein Structure Initiative of the NIH (U54GM094597 to L.T.). The Orchestra High Performance Compute Cluster at Harvard Medical School is a shared facility partly supported by NIH grant NCRR 1S10RR028832-01. T.W. is an investigator with the Howard Hughes Medical Institute.

Author information

Author notes

  1. Yu-Shan Hsiao & Chi-Yuan Chou

    Present address: Present addresses: Department of Otolaryngology, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114, USA (Y.-S.H.); Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan (C.-Y.C.).,

Authors and Affiliations

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

    Timothy H. Tran, Jeanyoung Jo, Chi-Yuan Chou, Lars E. P. Dietrich & Liang Tong

  2. Department of Cell Biology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Yu-Shan Hsiao & Thomas Walz

  3. Howard Hughes Medical Institute, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Thomas Walz

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Contributions

T.H.T. and C.-Y.C. performed cloning, protein expression, purification and crystallization experiments. T.H.T. and L.T. performed the crystallography experiments and calculation. Y.-S.H. and T.W. conducted the electron microscopy experiments. T.H.T. and C.-Y.C. performed the kinetic assays. J.J. and L.E.P.D. conducted the P. aeruginosa experiments. T.H.T., L.E.P.D., T.W. and L.T. wrote the paper.

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Correspondence to Liang Tong.

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Extended data figures and tables

Extended Data Figure 1 Domain organization of biotin-dependent carboxylases.

a, Reactions catalysed by biotin-dependent carboxylases. Biotin is linked to the side chain of a Lys residue in BCCP, and this flexible arm has a maximum length of ∼16 Å. The BCCP domain must also translocate to reach both active sites, separated by distances of 40–85 Å based on known holoenzyme structures (swinging domain model). b, Domain organizations of several representative biotin-dependent carboxylases. Homologous domains are given the same colours. The CT domain of PC has a completely different sequence and structure from those of ACC and PCC. The proteins are drawn to scale, and a scale bar is shown at the bottom. BT, BC–CT interaction domain; PT, PC tetramerization domain, also known as allosteric domain. c, Chemical structures of the substrates of ACC, PCC, LCC, MCC and GCC. The site of carboxylation is indicated with the red arrow.

Extended Data Figure 2 Phylogenetic trees for selected biotin-dependent carboxylases.

a, Phylogenetic tree for LCC homologues in a collection of organisms. The three homologues studied in this paper are shown in red. b, Phylogenetic tree for PCC homologues in a collection of organisms, based on a sequence alignment of the β subunit. Modified from an output from the Phylogeny.fr server44.

Extended Data Figure 3 Sequence alignment of long-chain acyl-CoA carboxylases (LCCs) from M. avium subspecies paratuberculosis (MapLCC), R. palustris (RpLCC), and P. aeruginosa (PaLCC).

The various domains in the proteins are labelled. The BCCP domain has two linkers to the rest of the protein. Modified from an output from ESPript45.

Extended Data Figure 4 Structural comparisons of domains in LCC with related enzymes.

a, Stereo drawing of the overlay of the structure of the BC domain dimer of MapLCC (in colour) with that of BC subunit dimer of E. coli ACC (in grey)20. The bound positions of biotin (black) and ADP (green) in the E. coli BC structure are also shown. The two-fold axis of the dimer is indicated by the black oval. With the two monomers at the bottom overlaid, a difference of 21° in the orientations of the two monomers at the top is observed. Most of the B domain of BC is ordered in one of the two monomers of MapLCC. In the other monomer, only weak electron density is observed for a few segments, and the B domain is not modelled. b, Overlay of the structures of the CT domain hexamer of MapLCC (in colour) and the β subunit of PCC (in grey)10. Each enzyme is highly conserved across species; the overlay should therefore be meaningful. c, Stereo drawing of the overlay of the CT domain dimer of MapLCC (in colour) and the β subunit of PCC (in grey). The view is down the red arrow in b. The bound position of biotin in the holoenzyme is shown in black. The position of CoA is modelled on that of CoA bound in the active site of the CT domain of yeast ACC29. d, Plot of the temperature factor value of each Cα atom in the two monomers (in red and blue). Several linker regions with high temperature factor values are indicated.

Extended Data Figure 5 Electron microscopy studies of LCC.

a, Representative raw image of negatively stained MapLCC. ‘S’ marks a side view of the holoenzyme, and ‘C’ indicates a contaminant. Scale bar, 500 Å. b, The 308 class averages of negatively stained MapLCC obtained from 19 generations of the iterative stable alignment and clustering (ISAC) procedure40 implemented in SPARX21. These class averages represent 65% (15,932 particles) of the entire data set (24,535 particles). Averages representing side views are marked with ‘S’, averages that were used to create Supplementary Video 1 are marked with an asterisk, and averages that represent a contaminant are marked with ‘C’. The side length of the individual panels is 340 Å. c, The averages obtained by classifying all 24,535 particles of negatively stained MapLCC into 300 classes using K-means classification in SPIDER39. Averages are shown in rows, with the most populous class at the top left and the least populous class at the bottom right. The side length of the individual panels is 340 Å.

Extended Data Figure 6 The CT active site of LCC.

a, Stereo drawing of the overlay of the CT active site (cyan and yellow) of MapLCC with that of PCC (grey)10. The model of CoA was obtained from the structure of the complex with yeast ACC CT domain29. The α6 helix in the N domain of monomer 6 shows a more closed conformation (indicated by the red arrow) and clashes with the CoA model. There is also a clash with the adenine base of CoA. There may be a conformational change in this region of MapLCC for CoA binding. b, Molecular surface of the CT active-site region of MapLCC. The α6 helix in the N domain of monomer 6 was removed for a clearer view of the active site.

Extended Data Figure 7 Phenotypic differences between wild-type and LCC knockout (ΔPA14_46320) P. aeruginosa strains, revealed by a colorimetric assay that monitors the reduction of a tetrazolium dye.

The conditions were identified from a screen that sampled 1,920 different media (Biolog Inc.). Assays were performed twice in each medium for the wild-type (red and orange) and mutant (blue and cyan) strains. Shown are activity profiles for strains incubated with Gly-Pro as the sole carbon source (top panel) and Asp-Phe, Glu-Val and Met-Asp as the sole nitrogen source (bottom panels). For each panel, the horizontal axis is time (24 h), and the vertical axis is OmniLog signal27.

Extended Data Table 1 Kinetic parameters of R. palustris LCC towards various substrates
Full size table
Extended Data Table 2 Data collection and refinement statistics
Full size table
Extended Data Table 3 Primers used for making the LCC deletion mutant
Full size table

Supplementary information

Variability in the architecture of the MapLCC holoenzyme

This animated GIF file illustrates the structural variability that results from the flexible tethering of the peripheral BC domains to the core formed by the CT domains. (MOV 756 kb)

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Tran, T., Hsiao, YS., Jo, J. et al. Structure and function of a single-chain, multi-domain long-chain acyl-CoA carboxylase. Nature 518, 120–124 (2015). https://doi.org/10.1038/nature13912

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