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^-1 mM^-1). 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 ₅₀¹⁵ 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.

Keywords: Agrobacterium tumefaciens; B factor; Glucuronic acid; Neutral drift; Thermostability; Uronate dehydrogenase.

Fig. 1

T₅₀¹⁵ value of WT and 17 variants of UdhAt. Variants containing the mutation H236K are marked dark grey. The solid line indicates the T₅₀¹⁵ value of WT (59 °C). Error bars are the standard deviation of four independent measurements

Fig. 2

Differential thermodynamic stability of single and triple variants of UdhAt in comparison to WT using GdmCl as denaturing agent. The single variant and all other variants containing the mutation H236K are marked dark grey

Fig. 3

Fluorescence intensity of WT, the best triple variant A41P/H101Y/H236K and the single variant H236K. All data points are the mean value of twenty samples with a standard deviation of <0.5

Fig. 4

Three monomers of the UdhAt with a close-up of one highlighted with the mutated amino acids in red, the product d-galactaro-1,5-lactone in green and the cofactor NADH in yellow