PhD defence by Kasper Hinz
MSBP1: A Key Regulator of Cytochrome P450 Dynamics
Plants have been utilized in our society for thousands of years due to their remarkable ability to produce a vast array of bioactive compounds, widely used as nutraceuticals and in pharmaceuticals. The ability to produce bioactive compounds has been developed through evolution as a key mechanism to adapt to changing environments. The biosynthesis of these compounds is highly fine-tuned, relying on an intricate network of metabolic processes that requires the organization of enzymes into functional enzyme complexes. One of the major enzyme families involved in the biosynthesis of specialized metabolites are the cytochrome P450 enzymes (CYPs). Anchored to the endoplasmic reticulum (ER), CYPs catalyze a broad variety of chemical reactions, driven by electron transfer from co-localized cytochrome P450 reductases (PORs). Although CYPs interact with PORs in a 1:1 stoichiometry, they outnumber PORs by a molar ratio of approximately 20:1. This imbalance implies the presence of a higherorder organization to maintain efficient CYP catalytic activity. Scaffold proteins are increasingly recognized as key regulators in various plant biosynthetic pathways. Notably, two membrane steroid-binding proteins (MSBPs) from Arabidopsis thaliana have been shown function as scaffold proteins of the lignin biosynthesis by regulating the stability and activity of three CYPs.
This PhD thesis explores the molecular function of MSBP1 from Sorghum bicolor, previously observed to co-purify with the biosynthetic enzymes of the dhurrin pathway (CYP79A1, CYP71E1, UGT85B1 and POR). We find that MSBP1 is upregulated in etiolated sorghum seedling upon treatment with either salt or jasmonic acids. Based on a weighted gene coexpression network analysis (WGCNA), we identify that MSBP1 clusters with proteins associated with ER remodeling and vesicle transport. Biochemical assays demonstrate that MSBP1 binds heme in vitro which facilitates dimerization of the protein. Additionally, overexpression of MSBP1 in sorghum protoplasts induces the formation of vesicles derived from the ER.
Reconstitution of the dhurrin pathway along with MSBP1 and sorghum POR isoforms (POR2a-c) or the A. thaliana POR (ATR1) in Saccharomyces cerevisiae, shows that MSBP1 increases the protein levels of the two CYPs 3-4 fold, regardless of the POR isoform.
Interestingly, dhurrin production increases twofold but only in the presence of POR2a and POR2b, indicating that MSBP1 selective enhances CYP activity in coordination with specific POR isoforms. Moreover, expression of a truncated MSBP1 variant lacking the disordered Cterminal (MSBP1ΔC), further increases the dhurrin production in yeast. Additionally, the function of MSBP1 and MSBP1ΔC is versatile as they facilitate increased production whenexpressed with the biosynthetic pathway of betanin in yeast.
Finally, the functinal mechanism of MSBP1 is discussed. We propose a model where MSBP1 plays a role in organizing CYPs and PORs within the ER membrane and potentially function by retaining CYPs in the ER membrane layer and prevent degradation.
Supervisors
Professor Birger Lindberg Møller, PLEN
Associate Professor Tomas Laursen, PLEN (co-supervisor)
Mette Sørensen (co-supervisor, prev PLEN)
Assessment committee
Principal Scientific Lead Chang-Jun Liu, Brookhaven National Laboratory
Research Professor Verena Siewers, Chalmers University of Technology
Professor Meike Burow, PLEN (Chair)