Biology subjects

Raytek, L. M.

Publications and source records attributed to Raytek, L. M..

3 recordsLinked to original sources

bioRxiv2025

Discovery of pseudobaptigenin synthase, completing the (-)-maackiain biosynthetic pathway

Pterocarpans are structurally complex defence compounds produced by legumes (Fabaceae). They are commonly associated with antimicrobial activity and thought to be synthesized de novo or accumulated in response to microbial pathogens. (-)-Maackiain is a lineage-specific pterocarpan detected in some legumes, including red clover (Trifolium pratense). The biosynthesis of (-)-maackiain involves a distinctive methylenedioxy bridge formation step, predicted to be catalyzed by a cytochrome P450. Specifically, this elusive P450 catalyzes the conversion of calycosin to pseudobaptigenin. We integrated metabolomic and transcriptomic datasets of red clover roots treated with the fungi, Fusarium oxysporum and Phoma medicaginis, to identify candidate P450 genes. Over 40 molecular features were characterized as (iso)flavonoid structures, including the highly abundant O-methylated isoflavones (formononetin and biochanin A), as well as their derivatives. Long infection with P. medicaginis resulted in significant increases in (-)-maackiain, trifolirhizin and other pterocarpans. Concurrently, fungal infections led to upregulation of core and specialized metabolism-related transcripts, including those encoding phenylpropanoid and (iso)flavonoid biosynthetic enzymes. Using weighted gene co-expression network analysis (WGCNA), variance-stabilized expression patterns, and enzyme-class phylogeny, we were able to curate five candidate cytochrome P450s for pseudobaptigenin synthase (PbS) activity, assayed in engineered yeast (Saccharomyces cerevisiae). One candidate P450 was capable of methylenedioxy bridge formation, converting calycosin to pseudobaptigenin and pratensein to 5-hydroxypseudobaptigenin. Therefore, it was renamed T. pratense pseudobaptigenin synthase (TpPbS/CYP76F319). The discovery of TpPbS facilitates the reconstruction of the complete (-)-maackiain biosynthetic pathway and the production of this pterocarpan chemistry at scale for health and agricultural applications. Significance statementO_LIThe discovery of pseudobaptigenin synthase in red clover (CYP76F319), a P450 that catalyzes the formation of a methylenedioxy bridge to convert calycosin to pseudobaptigenin and pratensein to 5-hydroxypseudobaptigenin. C_LIO_LIThe identification of enriched (iso)flavonoids and associated transcriptomic changes in red clover roots in response to two fungi with distinct infection lifestyles (hemibiotrophy and necrotrophy). C_LI

plant biology↗

bioRxiv2025

Chalcone isomerase-like impedes the lactone shunt and enhances flux partitioning in a bifurcated pathway towards isoflavonoid biosynthesis

The reconstitution of biosynthetic pathways in heterologous hosts is often challenged by the switch to a foreign cellular environment, lacking compatible structural or regulatory features. Auxiliary or non-catalytic proteins can play a critical role in modulating metabolic flux and pathway efficiency. Chalcone isomerase-like (CHIL) is a non-catalytic protein known to serve as a partner to chalcone synthase (CHS) in flavonoid biosynthesis, rectifying its promiscuous activity and preventing by-product formation, such as the aberrant p-coumaroyltriacetic acid lactone (CTAL). Here, we extended the characterization of CHILs to the legume-characteristic isoflavonoid pathway. We assessed four CHIL orthologs from diverse plant lineages: Glycine max (GmCHIL), Oryza sativa (OsCHIL), Selaginella moellendorffii (SmCHIL), and Marchantia polymorpha (MpCHIL). Structural modelling suggested that naringenin (flavanone) entry into the CHIL binding cleft may be sterically hindered compared to catalytic CHIs. Moreover, legume CHIL isoforms possess an additional bulky residue, Tyr48, that is expected to impose further constraints on ligand binding. In vitro, CHS produced up to 60% lactone CTAL instead of its desired output; however, CHIL suppressed this aberrant activity to 10%, concomitantly increasing target compound titers. Combinatorial enzyme and yeast biotransformation assays revealed a critical role for CHIL in conducting flux through chalcone, flavanone, and isoflavone biosynthesis. The inclusion of CHIL in our engineered yeast strains enhanced overall titers and, unexpectedly, promoted carbon flux toward the so-called deoxy-branch (isoliquiritigenin, liquiritigenin, and daidzein) by up to 67%, with a 33% increase in final daidzein titers. By extending CHIL characterization to the isoflavonoid pathway, we have revealed an expanded role for this auxiliary protein and underscored its utility in engineered metabolic contexts. Our findings reiterate the often-overlooked impact of non-catalytic proteins in shaping specialized metabolism. HIGHLIGHTSO_LICombinatorial enzyme assays reveal a species-dependent preference for CHILs from more closely related plant phyla by soybean CHS, which improved chalcone and downstream flavanone output by suppressing aberrant lactone formation. C_LIO_LIYeast co-expressing CHIL with the components of the isoflavonoid metabolon exhibited a 67% increase in flux through a legume-characteristic branch of the pathway, resulting in a 33% increase in titers of the major isoflavone, daidzein. C_LIO_LICHIL proteins can be included to engineer the output of phenylpropanoid-derived intermediates preferentially toward isoflavonoid biosynthesis. C_LIO_LIAuxiliary components, such as CHIL, can be designed to refine metabolic composition in bifurcated pathways, as well as enhancing general flux through pathways. C_LI

synthetic biology↗

bioRxiv2024

Heterologous expression and purification of glutamate decarboxylase-1 from the model plant Arabidopsis thaliana - characterization of the enzyme's in vitro truncation by thiol endopeptidase activity

Plant glutamate decarboxylase (GAD) is a Ca2+-calmodulin activated cytosolic enzyme that produces {gamma}-aminobutyrate (GABA) as the first committed step of the GABA shunt. This pathway circumvents the 2-oxoglutarate to succinate reactions of the mitochondrial tricarboxylic acid cycle. Our prior research established that in vivo phosphorylation of the root-specific AtGAD1 isozyme (AT5G17330) occurs at multiple N-terminal serine residues, following Pi resupply to Pi-starved cell cultures of the model plant Arabidopsis thaliana. The aim of the current investigation was to purify recombinant AtGAD1 following its expression in Escherichia coli to facilitate studies of the impact of site-specific phosphorylation on its kinetic properties. However, in vitro proteolytic truncation of a 5 kDa polypeptide from the C-terminus of 59 kDa AtGAD1 subunits occurred during its purification. Immunoblotting demonstrated that most protease inhibitors or cocktails that we tested were ineffective in suppressing partial AtGAD1 proteolysis during incubation of clarified extracts at 23 {degrees}C. Although the thiol modifiers N-ethylmaleimide or 2,2-dipyridyl disulfide negated AtGAD1 proteolysis, they also abolished its GAD activity. This indicates that an essential -SH group is needed for catalytic activity, and that AtGAD1 is susceptible to partial degradation either by an E. coli cysteine endopeptidase, or possibly via autoproteolytic activity. The inclusion of exogenous Ca2+/calmodulin in extraction and chromatography buffers facilitated the purification of non-proteolyzed AtGAD1 to a specific activity of 27 ({micro}mol GABA produced/mg) at optimal pH 5.8, while exhibiting an approximate 3-fold activation by Ca2+/CaM at pH 7.3. By contrast, the purified partially proteolyzed His6-AtGAD1 was >40% less active at both pH values, and only activated 2-fold by Ca2+/CaM at pH 7.3. These results emphasize the need to diagnose and prevent unwanted proteolysis before conducting kinetic studies of purified regulatory enzymes.

biochemistry↗