Structure-based catalytic optimization of a type III Rubisco from a hyperthermophile.

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• The Calvin-Benson-Bassham cycle utilizes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) for carbon dioxide fixation.
• Rubisco from Thermococcus kodakarensis (Tk-Rubisco) is a type III enzyme with high activity at elevated temperatures.
• Previous studies indicated that specific modifications to Tk-Rubisco's alpha-helix 6 can alter its catalytic performance at mesophilic temperatures.

Purpose of the Study:

• To elucidate the structural basis for altered catalytic activity of Tk-Rubisco mutants at mesophilic temperatures.
• To investigate the relationship between the flexibility of alpha-helix 6 and loop 6 regions and Tk-Rubisco's enzymatic activity.
• To engineer a Tk-Rubisco mutant with enhanced catalytic activity at ambient temperatures.

Main Methods:

• Determined crystal structures of wild-type Tk-Rubisco and SP4 and SP6 mutants.
• Analyzed structural differences, focusing on alpha-helix 6 movement and temperature factors.
• Constructed and characterized a new mutant (SP5-V330T) based on structural insights.

Main Results:

• Structural comparisons revealed movement and increased temperature factors in alpha-helix 6 across mutants.
• The SP6 mutant, with a full alpha-helix 6 replacement, showed improved catalytic performance at mesophilic temperatures.
• The novel SP5-V330T mutant, designed for increased flexibility, exhibited the highest activity among all tested mutants, albeit with reduced thermostability.

Conclusions:

• Increased flexibility in the alpha-helix 6 and loop 6 regions is crucial for enhancing Tk-Rubisco's catalytic activity at ambient temperatures.
• Structural modifications can decouple high activity at ambient temperatures from high thermostability.
• The SP5-V330T mutant represents a significant advancement in engineering Tk-Rubisco for improved performance under mesophilic conditions.