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Control of carotenoid biosynthesis through a heme-based cis-trans isomerase

Nature Chemical Biology volume 11, pages 598–605 (2015)Cite this article

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Abstract

Plants synthesize carotenoids, which are essential for plant development and survival. These metabolites also serve as essential nutrients for human health. The biosynthetic pathway for all plant carotenoids occurs in chloroplasts and other plastids and requires 15-cis-ζ-carotene isomerase (Z-ISO). It was not known whether Z-ISO catalyzes isomerization alone or in combination with other enzymes. Here we show that Z-ISO is a bona fide enzyme and integral membrane protein. Z-ISO independently catalyzes the cis-trans isomerization of the 15-15′ carbon-carbon double bond in 9,15,9′-cis-ζ-carotene to produce the substrate required by the subsequent biosynthetic-pathway enzyme. We discovered that isomerization depends upon a ferrous heme b cofactor that undergoes redox-regulated ligand switching between the heme iron and alternate Z-ISO amino acid residues. Heme b–dependent isomerization of a large hydrophobic compound in a membrane was previously undescribed. As an isomerase, Z-ISO represents a new prototype for heme b proteins and potentially uses a new chemical mechanism.

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Figure 1: Z-ISO is an isomerase and integral membrane protein localized to chloroplasts.
Figure 2: Z-ISO contains heme iron.
Figure 3: EPR shows multiple heme species.
Figure 4: MCD reveals redox-dependent changes in ligand coordination.
Figure 5: Testing mutation of putative heme ligands on enzyme activity, heme binding and UV-vis spectrum of Z-ISO.
Figure 6: Proposed mechanism of ligand rearrangement leading to active isomerization.

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Acknowledgements

We thank W. Hendrickson (Columbia University and New York Consortium on Membrane Protein Structure) for helpful discussions and use of the New York Structural Biology Center facilities. We thank M. Inouye (University of Medicine and Dentistry of New Jersey) for valuable advice on codon optimization and L. Bradbury (Lehman College and CUNY) for helpful discussions. E.T.W. was funded by the US National Institutes of Health (grant GM081160), CUNY and Lehman College. E.T.W. was also supported by travel funds from the Gordon Research Conferences to attend the Metals in Biology Gordon Research Conference, where E.T.W. was able to initiate collaborations with heme experts J.P. Hosler, A. Liu and J.H. Dawson through much-appreciated help from H. Gray (California Institute of Technology). The New York Consortium on Membrane protein structure (B.K. and J.D.L.) was supported through funds obtained from the US National Institutes of General Medical Sciences Protein Structure Initiative (PSI) program (grant GM095315). J.H.D. was funded by the US National Institutes of Health grant GM 26730; A.L. was funded by US National Institutes of Health grant R01GM108988 and the Georgia Research Alliance Distinguished Scholar Program. C.A.-D. was funded by The New Zealand Institute for Plant and Food Research Limited, New Zealand.

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Author notes

  1. Jiafeng Geng, Charles Ampomah-Dwamena & James D Love

    Present address: Present addresses: School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia, USA (J.G.), New Zealand Institute for Plant and Food Research Limited, Auckland, New Zealand (C.A.-D.) and Albert Einstein College of Medicine, Bronx, New York, USA (J.D.L.).,

Authors and Affiliations

  1. Department of Biological Sciences, Lehman College, City University of New York (CUNY), Bronx, New York, USA

    Jesús Beltrán, Maria Shumskaya, Charles Ampomah-Dwamena & Eleanore T Wurtzel

  2. Graduate School and University Center, CUNY, New York, New York, USA

    Jesús Beltrán & Eleanore T Wurtzel

  3. New York Structural Biology Center, New York, New York, USA

    Brian Kloss & James D Love

  4. Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi, USA

    Jonathan P Hosler

  5. Department of Chemistry, Georgia State University, Atlanta, Georgia, USA

    Jiafeng Geng & Aimin Liu

  6. Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina, USA

    Anuja Modi, John H Dawson & Masanori Sono

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Contributions

Wet-laboratory experiments were performed by J.B. (cloning, protein expression and purification, enzyme assays, HPLC, carotenoid analyses, mutagenesis, heme assays and UV-vis spectroscopy) and B.K. (cloning and protein expression), J.D.L. (design of pNYCOMPS vector, cloning, expression and protein-purification protocols) and J.P.H. (ICP-OES, UV-vis spectroscopy, heme assays and CO binding). EPR experiments were performed by J.G. and A.L. MCD experiments were performed by A.M., M. Sono and J.H.D. Localization and import, including related gene cloning, was conducted by M. Shumskaya. J.B. and E.T.W. performed bioinformatic analyses. C.A.-D. prepared mutant Z-ISO fusion proteins while on a short-term sabbatical in the Wurtzel laboratory. All authors contributed to data analysis and to the writing and editing of the manuscript.

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Correspondence to Eleanore T Wurtzel.

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Beltrán, J., Kloss, B., Hosler, J. et al. Control of carotenoid biosynthesis through a heme-based cis-trans isomerase. Nat Chem Biol 11, 598–605 (2015). https://doi.org/10.1038/nchembio.1840

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