Crystallization and X-ray diffraction analysis of a putative bacterial class I labdane-related diterpene synthase

Area of Science:

• Biochemistry
• Structural Biology
• Natural Products Chemistry

Background:

• Labdane-related diterpenoids are valuable natural products with pharmaceutical potential.
• These compounds are rarely found in bacterial species.
• Bacterial diterpene synthases are largely uncharacterized.

Purpose of the Study:

• To characterize a novel labdane-related diterpene synthase (LrdC) from a streptomycete.
• To determine the crystal structure of LrdC in complex with its cofactor and substrate analog.
• To elucidate the structural basis for labdane-related diterpenoid biosynthesis in bacteria.

Main Methods:

• Genome mining to identify the putative LrdC enzyme.
• Microbatch crystallization of the LrdC enzyme.
• X-ray diffraction analysis of LrdC crystals.
• Single-wavelength anomalous dispersion (SAD) phasing using an osmium derivative.

Main Results:

• Crystallization of LrdC in holo form (LrdC-Mg2+) and complex with inorganic pyrophosphate (LrdC-Mg2+-PPi).
• Native LrdC-Mg2+ crystals diffracted to 2.50 Å (space group P3221).
• LrdC-Mg2+-PPi crystals diffracted to 2.36 Å (space group P3221) and 2.57 Å (space group P21).
• The structure was determined using SAD phasing.

Conclusions:

• The crystal structure of LrdC provides insights into the catalytic mechanism of bacterial labdane-related diterpene biosynthesis.
• This work expands the known repertoire of bacterial natural product enzymes.
• The findings facilitate structure-based drug design for novel pharmaceutical compounds.