Thermostabilization of the uronate dehydrogenase from Agrobacterium tumefaciens by semi-rational design

Abstract

Aldaric acids represent biobased 'top value-added chemicals' that have the potential to substitute petroleum-derived chemicals. Until today they are mostly produced from corresponding aldoses using strong chemical oxidizing agents. An environmentally friendly and more selective process could be achieved by using natural resources such as seaweed or pectin as raw material. These contain large amounts of uronic acids as major constituents such as glucuronic acid and galacturonic acid which can be converted into the corresponding aldaric acids via an enzyme-based oxidation using uronate dehydrogenase (Udh). The Udh from Agrobacterium tumefaciens (UdhAt) features the highest catalytic efficiency of all characterized Udhs using glucuronic acid as substrate (829 s). Unfortunately, it suffers from poor thermostability. To overcome this limitation, we created more thermostable variants using semi-rational design. The amino acids for substitution were chosen according to the B factor in combination with four additional knowledge-based criteria. The triple variant A41P/H101Y/H236K showed higher kinetic and thermodynamic stability with a T 50 15 value of 62.2 °C (3.2 °C improvement) and a ∆∆G U of 2.3 kJ/mol compared to wild type. Interestingly, it was only obtained when including a neutral mutation in the combination.

Enhancing the thermostability and activity of uronate dehydrogenase from Agrobacterium tumefaciens LBA4404 by semi-rational engineering

“…In our study, we found that both thermostability and activity showed a marked improvement in the A41P/ H101Y/H236K UDH mutant derived from AtLBA4404. Similarly, a previously reported triple variant derived from AtC58, A41P/H101Y/H236K showed significantly improved thermostability; however, this mutant showed no enhancement in activity (Roth et al 2017). The reasons for this difference might be as follows: (a) although the UDH from A. tumefaciens LBA4404 is very similar to that from A. tumefaciens C58, it is not identical.…”

“…Identification of amino acid positions for mutation in At58 UDH was based on the structure-guided enzyme engineering and a previously report (Roth et al 2017) to select target residues in the UDH from AtLBA4404. Five residues (L36, A39, E79, H99 and H234) in the UDH amino acid sequence were identified as candidates and were altered by site-directed saturation mutagenesis (Fig.…”

“…However, its T 50 50 value is only 37 °C (Pick et al 2015). Subsequently, Roth et al achieved a more thermostable triple variant with a T 15 50 value of 62 °C and a ∆∆G U of 2.3 kJ/mol compared to wild-type (Roth et al 2017). However, this variant still does not meet the requirements of industry.…”

Biocatalysis and Strategies for Enzyme Improvement

“…SR design has a great potential to create specialized biocatalysts for the industrial use. For example, improved uronate dehydrogenase enzyme of high thermostability can be used to catalyze the production of glycaric acids (top-value chemical precursors for greener and less expensive biofuel synthesis) [110]. Nevertheless, even with reduced mutant libraries, SR approaches are not straight forward.…”

Enhancing the Thermostability of Rhizomucor miehei Lipase with a Limited Screening Library by Rational-Design Point Mutations and Disulfide Bonds

“…As another popular method, the B-factor iterative test (B-FIT), which rigidifies the most flexible residues in a protein, has been employed as a semirational strategy to improve the thermostability of many enzymes (4,(21)(22)(23)(24). Roth et al engineered uronate dehydrogenase from Agrobacterium tumefaciens by semirational design (25). The triple variant A41P/H101Y/H236K showed higher thermodynamic stability, with a T 50 15 value of 62.2°C (a 3.2°C improvement compared to the wild type [WT]).…”