Substrate-imprinted docking of Agrobacterium tumefaciens uronate dehydrogenase for increased substrate selectivity

Area of Science:

• Biochemistry and enzymology
• Protein engineering
• Metabolic engineering

Background:

• Agrobacterium tumefaciens uronate dehydrogenase (AtuUdh) is a short-chain dehydrogenase crucial for D-glucaric acid production.
• AtuUdh exhibits reversible, dual-substrate specificity for D-galacturonic and D-glucuronic acids.
• Understanding AtuUdh's substrate selectivity is key for optimizing its industrial applications.

Purpose of the Study:

• To engineer AtuUdh mutants with enhanced substrate selectivity and efficiency.
• To elucidate the structural basis for AtuUdh's substrate binding and catalytic mechanism.
• To improve the production of D-glucaric acid and D-galactaric acid.

Main Methods:

• Generated and screened 155 AtuUdh mutants, selecting 10 structurally stable variants.
• Employed a substrate-imprinted docking approach to predict mutant performance.
• Analyzed substrate and cofactor binding affinities for wild-type and mutant enzymes.

Main Results:

• Identified Q14F, S36L, and S75T mutants with high affinity for D-glucuronic acid and its intermediates.
• Discovered D34S, N112E, and S165E mutants exhibiting high selectivity for D-galacturonic acid production.
• Highlighted the roles of Ser75, Ser165, and Arg174 in substrate selectivity and transition state stabilization.

Conclusions:

• Engineered AtuUdh mutants demonstrate improved substrate specificity for targeted uronic acid production.
• The study provides a structural understanding of AtuUdh's mechanism, aiding future enzyme design.
• This work facilitates the development of more efficient biocatalysts for industrial applications.