About

Mary A. Schuler is a Professor of Cell & Developmental Biology and Biochemistry at the School of Molecular & Cellular Biology, Illinois College of Liberal Arts & Sciences. Her research focuses on molecular biology, biochemistry, and genomics, particularly pre-mRNA processing and P450 monooxygenases. Her work involves understanding the structure, function, and biochemical properties of plant and insect cytochrome P450 monooxygenases, which are membrane-bound proteins involved in critical metabolic pathways such as hydroxylations, epoxidations, and other complex reactions. Schuler's research aims to define the functions and structures of various P450s with roles in plant defense toxin production, phenylpropanoid metabolism, fatty acid modification, and insect detoxification processes. She employs a multidisciplinary approach combining structural modeling, biochemical analysis, genomics, and computational techniques to predict and validate P450 functions, substrate specificities, and gene regulation. Her projects include detailed studies on Arabidopsis thaliana P450s, elucidating their roles in stress responses and developmental processes, as well as investigations into insect P450s involved in detoxifying plant toxins and insecticides, with a focus on understanding the evolution of these enzymes and their contribution to insecticide resistance. Schuler's work integrates structural predictions, heterologous expression systems, and functional genomics to advance knowledge of P450 enzyme mechanisms and their applications in crop improvement and biopharmaceutical production.

Research topics

• Stereochemistry
• Chemistry
• Organic chemistry
• Biology
• Biochemistry
• Chromatography
• Botany

Selected publications

• Engineering C–C Bond Cleavage Activity into a P450 Monooxygenase Enzyme

  Journal of the American Chemical Society · 2023 · 28 citations

  • Chemistry
  • Stereochemistry
  • Organic chemistry

  The cytochrome P450 (CYP) superfamily of heme monooxygenases has demonstrated ability to facilitate hydroxylation, desaturation, sulfoxidation, epoxidation, heteroatom dealkylation, and carbon-carbon bond formation and cleavage (lyase) reactions. Seeking to study the carbon-carbon cleavage reaction of α-hydroxy ketones in mechanistic detail using a microbial P450, we synthesized α-hydroxy ketone probes based on the physiological substrate for a well-characterized benzoic acid metabolizing P450, CYP199A4. After observing low activity with wild-type CYP199A4, subsequent assays with an F182L mutant demonstrated enzyme-dependent C-C bond cleavage toward one of the α-hydroxy ketones. This C-C cleavage reaction was subject to an inverse kinetic solvent isotope effect analogous to that observed in the lyase activity of the human P450 CYP17A1, suggesting the involvement of a species earlier than Compound I in the catalytic cycle. Co-crystallization of F182L-CYP199A4 with this α-hydroxy ketone showed that the substrate bound in the active site with a preference for the (S)-enantiomer in a position which could mimic the topology of the lyase reaction in CYP17A1. Molecular dynamics simulations with an oxy-ferrous model of CYP199A4 revealed a displacement of the substrate to allow for oxygen binding and the formation of the lyase transition state proposed for CYP17A1. This demonstration that a correctly positioned α-hydroxy ketone substrate can realize lyase activity with an unusual inverse solvent isotope effect in an engineered microbial system opens the door for further detailed biophysical and structural characterization of CYP catalytic intermediates.

  PublisherOA PDFDOI

• Catalytic Site Constraints in the P450s Mediating Loganic Acid (7DLH) and Secologanic Acid Synthesis (SLAS) in <i>Camptotheca</i>

  Biochemistry · 2023-09-01 · 4 citations

  articleSenior authorCorresponding

  Terpene indole alkaloids (TIAs) are plant-derived natural products synthesized in low levels in medicinal plants such as Catharanthus roseus and Camptotheca acuminata. TIA pathways species utilize several CYP72A subfamily members to form loganic acid from 7-deoxyloganic acid (a simple hydroxylation) as well as secologanin and secologanic acid from loganin and loganic acid (a C–C bond scission). Divergences in the specificities of these P450s have allowed Camptotheca secologanic acid synthases (SLASs) to become bifunctional enzymes capable of performing both reactions. In contrast, Catharanthus 7-deoxyloganic acid hydroxylase (7DLH) and secologanin synthase (SLS) have remained monofunctional enzymes capable either of monooxygenation or C–C bond scission. Our in vitro reconstitutions have now demonstrated that Camptotheca also contains a monofunctional 7DLH capable only of hydroxylating 7-deoxyloganic acid. Mutageneses aimed at evaluating residues important for the tight specificity of Camptotheca 7DLH (CYP72A729) and the broad specificity of SLAS (CYP72A564) have identified several residues where reciprocal switches substantially affect their activities: Lys128His in 7DLH increases hydroxylation of 7-deoxyloganic acid, and His132Lys in SLAS decreases this hydroxylation and C–C bond scissions of loganic acid and loganin; Gly321Ser in 7DLH does not affect hydroxylation of 7-deoxyloganic acid, whereas Ser324Gly in SLAS significantly increases C–C bond scission of loganic acid; Asp332Glu in the acid–alcohol pair of 7DLH increases hydroxylation of 7-deoxyloganic acid, whereas Glu335Asp in SLAS completely eliminates both of its activities. These mutations that enhance or eliminate these respective activities have significant potential to aid engineering efforts aimed at increasing TIA production in cell cultures, microbial systems, and/or other plants.

  PublisherDOI

• Single mutations toggle the substrate selectivity of multifunctional Camptotheca secologanic acid synthases

  Journal of Biological Chemistry · 2022 · 9 citations

  Senior authorCorresponding
  • Biochemistry
  • Chemistry
  • Biology

  Terpene indole alkaloids (TIAs) are plant-derived specialized metabolites with widespread use in medicine. Species-specific pathways derive various TIAs from common intermediates, strictosidine or strictosidinic acid, produced by coupling tryptamine with secologanin or secologanic acid. The penultimate reaction in this pathway is catalyzed by either secologanin synthase (SLS) or secologanic acid synthase (SLAS) according to whether plants produce secologanin from loganin or secologanic acid from loganic acid. Previous work has identified SLSs and SLASs from different species, but the determinants of selectivity remain unclear. Here, combining molecular modeling, ancestral sequence reconstruction, and biochemical methodologies, we identified key residues that toggle SLS and SLAS selectivity in two CYP72A (cytochrome P450) subfamily enzymes from Camptotheca acuminata. We found that the positions of foremost importance are in substrate recognition sequence 1 (SRS1), where mutations to either of two adjacent histidine residues switched selectivity; His131Phe selects for and increases secologanin production whereas His132Asp selects for secologanic acid production. Furthermore, a change in SRS3 in the predicted substrate entry channel (Arg/Lys270Thr) and another in SRS4 at the start of the I-helix (Ser324Glu) decreased enzyme activity toward either substrate. We propose that the Camptotheca SLASs have maintained the broadened activities found in a common asterid ancestor, even as the Camptotheca lineage lost its ability to produce loganin while the campanulid and lamiid lineages specialized to produce secologanin by acquiring mutations in SRS1. The identification here of the residues essential for the broad substrate scope of SLASs presents opportunities for more tailored heterologous production of TIAs.

  PublisherOA PDFDOI

• P450 variations bifurcate the early terpene indole alkaloid pathway in Catharanthus roseus and Camptotheca acuminata

  Phytochemistry · 2021 · 14 citations

  Senior authorCorresponding
  • Chemistry
  • Biochemistry
  • Stereochemistry
  PublisherOA PDFDOI

• P 450 variations bifurcate the early terpene indole alkaloid pathway in Catharanthus roseus and Camptotheca acuminata

  Global Biodiversity Information Facility · 2021-01-01

  datasetOpen accessSenior author

  This dataset contains the digitized treatments in Plazi based on the original journal article Miller, Justin C., Hollatz, Allison J., Schuler, Mary A. (2021): P 450 variations bifurcate the early terpene indole alkaloid pathway in Catharanthus roseus and Camptotheca acuminata. Phytochemistry (112626) 183: 1-13, DOI: 10.1016/j.phytochem.2020.112626, URL: http://dx.doi.org/10.1016/j.phytochem.2020.112626

  PublisherDOI

• Substrate‐specificity of cytochrome P450‐mediated detoxification as an evolutionary strategy for specialization on furanocoumarin‐containing hostplants: CYP6AE89 in parsnip webworms

  Insect Molecular Biology · 2019-08-08 · 37 citations

  article

  The parsnip webworm, Depressaria pastinacella, is restricted to two hostplant genera containing six structurally diverse furanocoumarins. Of these, imperatorin is detoxified by a specialized cytochrome P450, CYP6AB3. A previous whole-larva transcriptome analysis confirmed the presence of nine transcripts that belong to the CYP6AE subfamily. Here, by examining midgut-specific gene expression patterns we determined that CYP6AE89 transcripts were highly expressed and furanocoumarin-inducible. Computer docking and energy-minimization of a CYP6AE89 model with all six furanocoumarins showed that 5-methoxylated bergapten and 8-methoxylated xanthotoxin had the smallest distances from the heme to the proton-donor residue in the catalytic I-helix, and that the 5,8-dimethoxylated isopimpinellin and bergapten had the smallest energy-minimized distance from the heme oxygen to the furan ring double bond. To evaluate this prediction, we expressed the CYP6AE89 protein in an Escherichia coli system, and used it to detect high catalytic activity against the two mono-methoxylated linear furanocoumarins - bergapten and xanthotoxin - and weak activity against isopimpinellin. Thus, CYP6AE89, like CYP6AB3, is probably specialized for detoxifying only a subset of hostplant furanocoumarins. A maximum-likelihood tree built with six representative lepidopterans with manually annotated cytochrome P450s shows that CYP6AE89 may have evolved much faster than the other CYP6AE proteins, possibly indicative of host selection pressure.

  PublisherDOI

• Nanodiscs as a New Tool to Examine Lipid–Protein Interactions

  Methods in molecular biology · 2019-01-01 · 28 citations

  review
  PublisherDOI

• Allele-defined genome of the autopolyploid sugarcane Saccharum spontaneum L.

  Nature Genetics · 2018-10-08 · 787 citations

  articleOpen access

  Modern sugarcanes are polyploid interspecific hybrids, combining high sugar content from Saccharum officinarum with hardiness, disease resistance and ratooning of Saccharum spontaneum. Sequencing of a haploid S. spontaneum, AP85-441, facilitated the assembly of 32 pseudo-chromosomes comprising 8 homologous groups of 4 members each, bearing 35,525 genes with alleles defined. The reduction of basic chromosome number from 10 to 8 in S. spontaneum was caused by fissions of 2 ancestral chromosomes followed by translocations to 4 chromosomes. Surprisingly, 80% of nucleotide binding site-encoding genes associated with disease resistance are located in 4 rearranged chromosomes and 51% of those in rearranged regions. Resequencing of 64 S. spontaneum genomes identified balancing selection in rearranged regions, maintaining their diversity. Introgressed S. spontaneum chromosomes in modern sugarcanes are randomly distributed in AP85-441 genome, indicating random recombination among homologs in different S. spontaneum accessions. The allele-defined Saccharum genome offers new knowledge and resources to accelerate sugarcane improvement.

  PublisherOA PDFDOI

• Pastinaca sativa P450s - CYP71AJ4 variants in New Zealand and North America

  2018-01-01

  articleOpen accessSenior author

  Nucleotide sequences from wild parsnip CYP71AJ4 (angelic in synthase. Genbank EF191021) were obtained by Sanger sequencing. Seeds from individual plants from different populations were harvested to obtain corresponding cDNA. The cDNA was cloned and directly sequenced. Aminoacid translations were obtained using standard codon usage. Alignments of CYP71AJ4 sequences (involved in angular furanocoumarin biosynthesis) with as the reference sequence. Consistent amino acid variabilities were found between some populations. The relationship between sequencing variability and selective pressure is not yet known.

  PublisherOA PDFDOI

• Publisher Correction: Allele-defined genome of the autopolyploid sugarcane Saccharum spontaneum L

  Nature Genetics · 2018-11-13 · 18 citations

  erratumOpen access
  PublisherOA PDFDOI

Recent grants

• NIH Grant R01GM039025

  NIH · $1.7M · 1998

• NIH Grant R01GM079530

  NIH · $2.1M · 2013

• NIH Grant R01GM071826

  NIH · $1.2M · 2009

• Arabidopsis 2010: Functional Genomics of Arabidopsis P450s

  NSF · $3.5M · 2001–2006

• NIH Grant R01GM050007

  NIH · $1.8M · 2004

Frequent coauthors

• May R. Berenbaum

  University of Illinois Urbana-Champaign

  45 shared

• Todd P. Michael

  Salk Institute for Biological Studies

  28 shared

• Matthew E. Hudson

  University of Illinois Urbana-Champaign

  28 shared

• Danièle Werck‐Reichhart

  Centre National de la Recherche Scientifique

  25 shared

• Steve A. Kay

  University of Southern California

  25 shared

• Yinghong Pan

  25 shared

• Joanne Chory

  Salk Institute for Biological Studies

  25 shared

• David R. Nelson

  University of Tennessee Health Science Center

  22 shared