De novo Sequencing and Native Mass Spectrometry Reveals Hetero-Association of Dirigent Protein Homologs and Potential Interacting Proteins in Forsythia × intermedia Mowei Zhou,1 * Joseph A. Laureanti,2 Callum J. Bell,3 Mi Kwon,4 Qingyan Meng,4 Irina V. Novikova,1 Dennis G. Thomas,5 Carrie D. Nicora,5 Ryan L. Sontag,5 Diana L. Bedgar,4 Isabelle O’Bryon,6 Eric D. Merkley,6 Bojana Ginovska,2 John R. Cort,4,5 Laurence B. Davin, and 4 Norman G. Lewis4 1. Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, USA 2. Physical and Computational Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA, USA 3. National Center for Genome Resources, Santa Fe, New Mexico, USA 4. Institute of Biological Chemistry, Washington State University, Pullman, WA, USA 5. Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, USA 6. National Security Division, Pacific Northwest National Laboratory, Richland, Washington, USA Corresponding email:
[email protected] Abstract: The discovery of dirigent proteins (DPs) and their functions in plant phenol biochemistry was made over two decades ago with Forsythia × intermedia. Stereo-selective, DP-guided, monolignol-derived radical coupling in vitro was then reported to afford the optically active lignan, (+)-pinoresinol from coniferyl alcohol, provided one-electron oxidase/oxidant capacity was present. It later became evident that DPs have several distinct sub-families. In vascular plants, DPs hypothetically function, along with other essential enzymes/proteins (e.g. oxidases), as part of lignin/lignan forming complexes (LFCs). Herein, we used an integrated bottom-up, top-down, and native mass spectrometry approach to detect potential interacting proteins in a DP-enriched solubilized protein fraction from Forsythia × intermedia, via adaptation of our initial report of DP solubilization and purification.