Structure-based engineering of strictosidine synthase: auxiliary for alkaloid libraries

Area of Science:

• Biochemistry
• Enzymology
• Plant Science

Background:

• Strictosidine synthase (STR1) is crucial for synthesizing over 2000 monoterpenoid indole alkaloids via the Pictet-Spengler reaction.
• Understanding STR1's structure and function is key to modulating alkaloid production in plants.

Purpose of the Study:

• To elucidate the crystal structure of Rauvolfia serpentina strictosidine synthase (STR1) in complex with strictosidine.
• To rationally redesign STR1 through site-directed mutagenesis to alter its substrate acceptance.
• To explore the chemoenzymatic synthesis of novel alkaloid derivatives for pharmacological screening.

Main Methods:

• X-ray crystallography was used to determine the structure of STR1 bound to strictosidine.
• Site-directed mutagenesis was employed to generate the Val208Ala mutant of STR1.
• Enzyme kinetics and product analysis were performed to assess the impact of mutations on substrate acceptance and product formation.
• A chemoenzymatic approach combining STR1 activity with subsequent enzymatic steps was developed.

Main Results:

• The crystal structure of STR1-strictosidine complex provided insights into the enzyme's active site.
• The Val208Ala mutation in STR1 demonstrated altered substrate acceptance.
• The study successfully synthesized 10-methyl- and 10-methoxystrictosidines using the engineered STR1.
• A chemoenzymatic method was established for generating diverse alkaloid libraries.

Conclusions:

• Rational redesign of STR1 is feasible, enabling modulation of its substrate specificity.
• Engineered STR1 variants can produce novel alkaloid structures.
• The developed chemoenzymatic strategy offers a powerful platform for generating alkaloid libraries for drug discovery and pharmacological screening.