Engineering enhanced thermostability into the Geobacillus pallidus nitrile hydratase

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Structural Biology

Background:

• Nitrile hydratases (NHases) are crucial biocatalysts for converting nitriles to amides.
• Low thermostability limits NHase applications, hindering rational enzyme design.
• Understanding the structural basis of NHase thermostability is essential for improvement.

Purpose of the Study:

• To enhance the thermostability of a nitrile hydratase (NHase) from *Geobacillus pallidus*.
• To elucidate the structural determinants of increased thermostability in NHase mutants.
• To provide insights for rational enzyme design of thermostable NHases.

Main Methods:

• Random mutagenesis of the *Geobacillus pallidus* NHase gene.
• Selection and characterization of thermostable NHase mutants.
• High-resolution X-ray crystallography of wild-type and mutant NHases.

Main Results:

• Four NHase mutants exhibited 3- to 15-fold higher thermostability.
• Mutant 9E achieved a resolution of 1.15 Å, the highest for an NHase crystal structure.
• Structural analysis revealed enhanced thermostability due to increased salt bridges, hydrogen bonds, and stabilized secondary structures.
• Structured water molecules were identified as important mediators of hydrogen bonds, contributing to stability.

Conclusions:

• Rational enzyme design for NHase thermostability can be guided by structural insights.
• Enhanced intra- and inter-subunit interactions, including those involving structured water, are key to improving NHase stability.
• The identified structural modifications provide a foundation for engineering more robust NHases for industrial applications.