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Substrate-mediated electron transfer in peptidylglycine α-hydroxylating monooxygenase

Nature Structural Biology volume 6, pages 976–983 (1999)Cite this article

Abstract

Peptide amidation is a ubiquitous posttranslational modification of bioactive peptides. Peptidylglycine α-hydroxylating monooxygenase (PHM; EC 1.14.17.3), the enzymne that catalyzes the first step of this reaction, is composed of two domains, each of which binds one copper atom. The coppers are held 11 Å apart on either side of a solvent-filled interdomain cleft, and the PHM reaction requires electron transfer between these sites. A plausible mechanism for electron transfer might involve interdomain motion to decrease the distance between the copper atoms. Our experiments show that PHM catalytic core (PHMcc) is enzymatically active in the crystal phase, where interdomain motion is not possible. Instead, structures of two states relevant to catalysis indicate that water, substrate and active site residues may provide an electron transfer pathway that exists only during the PHM catalytic cycle.

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Figure 1: Reaction catalyzed by bifunctional PAM.
Figure 2: A representation of the PHMcc fold.
Figure 3: Schematic diagram of crystal activity experiment.
Figure 4: Reduced and oxidized PHMcc active sites.
Figure 5: Stereo view of the electron density connecting Gln 170 and substrate in ox-PHMcc–sub.
Figure 6: Superposition of reduced and oxidized PHMcc active sites.
Figure 7: Calculated electron transfer pathways.
Figure 8: Mechanism proposed for PHM involving electron transfer through the substrate.

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Acknowledgements

We thank C. Ogata and the staff at beamline X4A for assistance during data collection at the NSLS. Beamline X4A at the NSLS, a DOE facility, is supported by the Howard Hughes Medical Institute. We thank J. Berg, J. Wehrle and N. Carrasco for critical review of this manuscript. This work was supported by grants from the NIDDK to B.A.E and the NIGMS to L.M.A.

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  1. Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, 21205, Maryland, USA

    Sean T. Prigge & L. Mario Amzel

  2. Departments of Neuroscience and Physiology, Johns Hopkins School of Medicine, Baltimore, 21205, Maryland, USA

    Aparna S. Kolhekar, Betty A. Eipper & Richard E. Mains

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  1. Sean T. Prigge
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Correspondence to L. Mario Amzel.

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Prigge, S., Kolhekar, A., Eipper, B. et al. Substrate-mediated electron transfer in peptidylglycine α-hydroxylating monooxygenase. Nat Struct Mol Biol 6, 976–983 (1999). https://doi.org/10.1038/13351

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