Dirigent proteins (DPs) were first discovered over two decades ago from the plant Forsythia × intermedia. Later, other DPs were discovered, and sequence analysis suggested that DP homologs have several distinct sub-families. All known DPs mediate stereo-selective coupling of plant phenols. Recent studies have demonstrated the potential of DPs for highly efficient in vitro stereo-selective synthesis of natural product analogs not easily achievable with conventional chemical approaches. In vascular plants, DPs hypothetically function, along with other essential enzymes/proteins (e.g. oxidases), as part of lignin/lignan forming complexes (LFCs). However, very limited experimental data are available to elucidate the putative LFC. Part of the challenges is the lack of sequenced genome of the Forsythia plant where the original DP was purified from. Herein, we used de novo sequencing mass spectrometry to identify other proteins in a DP-enriched solubilized protein fraction, via adaptation of our initial report of DP solubilization and purification. With the help of transcriptome and genome data from homologous plants, we confidently identified 14 proteins in the fraction, including two new DP homologs not previously reported. Native mass spectrometry of the same fraction showed they also formed hetero-trimers. Molecular dynamics simulations suggest that similar hetero-trimers were possible between DP homologs with comparable sequence similarity. Other identified proteins in the DP-enriched preparation were putatively associated with DP function or the cell wall. Although their co-occurrence after extraction and chromatographic separation is suggestive for their associations in vivo, none were found to form long-lived complexes with DPs under the specific experimental conditions we have explored. We are currently developing new purification workflows to better capture the putative complexes. At the same time, we are expanding our study to other DP homologs in model plant organisms using our integrated mass spectrometry method. We believe our study will reveal novel molecular mechanisms of DP for understanding of biosynthesis of lignans and lignins.

Native mass spectrometry and de novo sequencing revealed hetero-complexes of new dirigent protein homologs in Forsythia x intermedia

ACS Spring 2022

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