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1.
J Mol Biol ; 436(11): 168586, 2024 Jun 01.
Article in English | MEDLINE | ID: mdl-38663544

ABSTRACT

Stabilizing proteins without otherwise hampering their function is a central task in protein engineering and design. PYR1 is a plant hormone receptor that has been engineered to bind diverse small molecule ligands. We sought a set of generalized mutations that would provide stability without affecting functionality for PYR1 variants with diverse ligand-binding capabilities. To do this we used a global multi-mutant analysis (GMMA) approach, which can identify substitutions that have stabilizing effects and do not lower function. GMMA has the added benefit of finding substitutions that are stabilizing in different sequence contexts and we hypothesized that applying GMMA to PYR1 with different functionalities would identify this set of generalized mutations. Indeed, conducting FACS and deep sequencing of libraries for PYR1 variants with two different functionalities and applying a GMMA analysis identified 5 substitutions that, when inserted into four PYR1 variants that each bind a unique ligand, provided an increase of 2-6 °C in thermal inactivation temperature and no decrease in functionality.


Subject(s)
DNA Mutational Analysis , Plant Growth Regulators , Plant Proteins , Protein Engineering , Protein Stability , Receptors, Cell Surface , Amino Acid Substitution/genetics , Ligands , Mutation , Protein Binding , Protein Engineering/methods , DNA Mutational Analysis/methods , Kluyveromyces , Plant Growth Regulators/chemistry , Plant Proteins/chemistry , Plant Proteins/genetics , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/genetics , Abscisic Acid/metabolism
2.
New Phytol ; 237(5): 1810-1825, 2023 03.
Article in English | MEDLINE | ID: mdl-36451537

ABSTRACT

Plant-specialized metabolism is complex, with frequent examples of highly branched biosynthetic pathways, and shared chemical intermediates. As such, many plant-specialized metabolic networks are poorly characterized. The N-methyl Δ1 -pyrrolinium cation is a simple pyrrolidine alkaloid and precursor of pharmacologically important tropane alkaloids. Silencing of pyrrolidine ketide synthase (AbPyKS) in the roots of Atropa belladonna (Deadly Nightshade) reduces tropane alkaloid abundance and causes high N-methyl Δ1 -pyrrolinium cation accumulation. The consequences of this metabolic shift on alkaloid metabolism are unknown. In this study, we utilized discovery metabolomics coupled with AbPyKS silencing to reveal major changes in the root alkaloid metabolome of A. belladonna. We discovered and annotated almost 40 pyrrolidine alkaloids that increase when AbPyKS activity is reduced. Suppression of phenyllactate biosynthesis, combined with metabolic engineering in planta, and chemical synthesis indicates several of these pyrrolidines share a core structure formed through the nonenzymatic Mannich-like decarboxylative condensation of the N-methyl Δ1 -pyrrolinium cation with 2-O-malonylphenyllactate. Decoration of this core scaffold through hydroxylation and glycosylation leads to mono- and dipyrrolidine alkaloid diversity. This study reveals the previously unknown complexity of the A. belladonna root metabolome and creates a foundation for future investigation into the biosynthesis, function, and potential utility of these novel alkaloids.


Subject(s)
Alkaloids , Atropa belladonna , Atropa belladonna/metabolism , Alkaloids/metabolism , Tropanes/chemistry , Tropanes/metabolism , Pyrrolidines/metabolism
3.
Nat Commun ; 13(1): 3832, 2022 07 02.
Article in English | MEDLINE | ID: mdl-35780230

ABSTRACT

Plant alkaloids constitute an important class of bioactive chemicals with applications in medicine and agriculture. However, the knowledge gap of the diversity and biosynthesis of phytoalkaloids prevents systematic advances in biotechnology for engineered production of these high-value compounds. In particular, the identification of cytochrome P450s driving the structural diversity of phytoalkaloids has remained challenging. Here, we use a combination of reverse genetics with discovery metabolomics and multivariate statistical analysis followed by in planta transient assays to investigate alkaloid diversity and functionally characterize two candidate cytochrome P450s genes from Atropa belladonna without a priori knowledge of their functions or information regarding the identities of key pathway intermediates. This approach uncovered a largely unexplored root localized alkaloid sub-network that relies on pseudotropine as precursor. The two cytochrome P450s catalyze N-demethylation and ring-hydroxylation reactions within the early steps in the biosynthesis of diverse N-demethylated modified tropane alkaloids.


Subject(s)
Alkaloids , Tropanes , Alkaloids/chemistry , Cytochrome P-450 Enzyme System/genetics , Metabolomics , Tropanes/metabolism
4.
ACS Synth Biol ; 8(3): 474-481, 2019 03 15.
Article in English | MEDLINE | ID: mdl-30721031

ABSTRACT

Enzymes are the ultimate entities responsible for chemical transformations in natural and engineered biosynthetic pathways. However, many natural enzymes suffer from suboptimal functional expression due to poor intrinsic protein stability. Further, stability enhancing mutations often come at the cost of impaired function. Here we demonstrate an automated protein engineering strategy for stabilizing enzymes while retaining catalytic function using deep mutational scanning coupled to multiple-filter based screening and combinatorial mutagenesis. We validated this strategy by improving the functional expression of a Type III polyketide synthase from the Atropa belladonna biosynthetic pathway for tropane alkaloids. The best variant had a total of 8 mutations with over 25-fold improved activity over wild-type in E. coli cell lysates, an improved melting temperature of 11.5 ± 0.6 °C, and only minimal reduction in catalytic efficiency. We show that the multiple-filter approach maintains acceptable sensitivity with homology modeling structures up to 4 Å RMS. Our results highlight an automated protein engineering tool for improving the stability and solubility of difficult to express enzymes, which has impact for biotechnological applications.


Subject(s)
Acyltransferases/chemistry , Acyltransferases/genetics , Atropa belladonna/enzymology , Biotechnology/methods , Data Science/methods , Protein Engineering/methods , Acyltransferases/metabolism , Belladonna Alkaloids/metabolism , Biosynthetic Pathways , Codon, Nonsense , Enzyme Stability/genetics , Escherichia coli/metabolism , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/metabolism , Luminescent Agents/chemistry , Luminescent Agents/metabolism , Mutagenesis , Mutation, Missense , Saccharomyces cerevisiae/metabolism , Solubility , Transition Temperature
5.
Nat Commun ; 9(1): 5281, 2018 12 11.
Article in English | MEDLINE | ID: mdl-30538251

ABSTRACT

Tropinone is the first intermediate in the biosynthesis of the pharmacologically important tropane alkaloids that possesses the 8-azabicyclo[3.2.1]octane core bicyclic structure that defines this alkaloid class. Chemical synthesis of tropinone was achieved in 1901 but the mechanism of tropinone biosynthesis has remained elusive. In this study, we identify a root-expressed type III polyketide synthase from Atropa belladonna (AbPYKS) that catalyzes the formation of 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid. This catalysis proceeds through a non-canonical mechanism that directly utilizes an unconjugated N-methyl-Δ1-pyrrolinium cation as the starter substrate for two rounds of malonyl-Coenzyme A mediated decarboxylative condensation. Subsequent formation of tropinone from 4-(1-methyl-2-pyrrolidinyl)-3-oxobutanoic acid is achieved through cytochrome P450-mediated catalysis by AbCYP82M3. Silencing of AbPYKS and AbCYP82M3 reduces tropane levels in A. belladonna. This study reveals the mechanism of tropinone biosynthesis, explains the in planta co-occurrence of pyrrolidines and tropanes, and demonstrates the feasibility of tropane engineering in a non-tropane producing plant.


Subject(s)
Atropa belladonna/enzymology , Atropa belladonna/metabolism , Cytochrome P-450 Enzyme System/metabolism , Plant Proteins/metabolism , Polyketide Synthases/metabolism , Tropanes/chemistry , Tropanes/metabolism , Atropa belladonna/genetics , Cyclization , Cytochrome P-450 Enzyme System/genetics , Molecular Structure , Plant Proteins/genetics , Plant Roots/chemistry , Plant Roots/enzymology , Plant Roots/genetics , Plant Roots/metabolism , Polyketide Synthases/genetics
6.
Plant Cell ; 26(9): 3745-62, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25228340

ABSTRACT

The tropane alkaloids, hyoscyamine and scopolamine, are medicinal compounds that are the active components of several therapeutics. Hyoscyamine and scopolamine are synthesized in the roots of specific genera of the Solanaceae in a multistep pathway that is only partially elucidated. To facilitate greater understanding of tropane alkaloid biosynthesis, a de novo transcriptome assembly was developed for Deadly Nightshade (Atropa belladonna). Littorine is a key intermediate in hyoscyamine and scopolamine biosynthesis that is produced by the condensation of tropine and phenyllactic acid. Phenyllactic acid is derived from phenylalanine via its transamination to phenylpyruvate, and mining of the transcriptome identified a phylogenetically distinct aromatic amino acid aminotransferase (ArAT), designated Ab-ArAT4, that is coexpressed with known tropane alkaloid biosynthesis genes in the roots of A. belladonna. Silencing of Ab-ArAT4 disrupted synthesis of hyoscyamine and scopolamine through reduction of phenyllactic acid levels. Recombinant Ab-ArAT4 preferentially catalyzes the first step in phenyllactic acid synthesis, the transamination of phenylalanine to phenylpyruvate. However, rather than utilizing the typical keto-acid cosubstrates, 2-oxoglutarate, pyruvate, and oxaloacetate, Ab-ArAT4 possesses strong substrate preference and highest activity with the aromatic keto-acid, 4-hydroxyphenylpyruvate. Thus, Ab-ArAT4 operates at the interface between primary and specialized metabolism, contributing to both tropane alkaloid biosynthesis and the direct conversion of phenylalanine to tyrosine.


Subject(s)
Atropa belladonna/enzymology , Biosynthetic Pathways , Phenylalanine/metabolism , Phenylpyruvic Acids/metabolism , Plant Roots/enzymology , Transaminases/metabolism , Tropanes/metabolism , Atropa belladonna/genetics , Biosynthetic Pathways/genetics , Computer Simulation , Gene Expression Regulation, Plant , Gene Silencing , Kinetics , Molecular Sequence Annotation , Molecular Sequence Data , Multigene Family , Phylogeny , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Analysis, RNA , Transaminases/genetics , Transcriptome/genetics , Tropanes/chemistry
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