Analysis of enzyme kinetics of fungal methionine synthases in an optimized colorimetric microscale assay for measuring cobalamin-independent methionine synthase activity

Aspergillus spp. and Rhizopus spp., used in solid-state plant food fermentations, encode cobalamin-independent methionine synthase activity (MetE, EC 2.1.1.14). Here, we examine the enzyme kinetics, reaction activation energies (Ea), thermal robustness, and structural folds of three MetEs from three different food-fermentation relevant fungi, Aspergillus sojae, Rhizopus delemar, and Rhizopus microsporus, and compare them to the MetE from Escherichia coli. We also downscaled and optimized a colorimetric assay to allow direct MetE activity measurements in microplates. The catalytic rates, k_cat, of the three fungal MetE enzymes on the methyl donor (6S)-5-methyl-tetrahydropteroyl-L-glutamate3 ranged from 1.2 to 3.3 min⁻¹ and KM values varied from 0.8 to 6.8 µM. The k_cat was lowest for the R. delemar MetE, but this enzyme also had the lowest K_M thus resulting in the highest k_cat/K_M of ∼1.4 min⁻¹ µM⁻¹ among the three fungal enzymes. The k_cat was higher for the E. coli enzyme, 12 min⁻¹, but K_M was 6.4 µM, resulting in k_cat/K_M of ∼1.9 min⁻¹ µM⁻¹. The E_a values of the fungal MetEs ranged from 52 to 97 kJ mole⁻¹ and were higher than that of the E. coli MetE (38.7 kJ mole⁻¹). The predicted structural folds of the MetEs were very similar. T_m values of the fungal MetEs ranged from 41 to 54 °C, highest for the A. sojae enzyme (54 °C), lowest for the R. delemar (41 °C). At 30 °C, the half-lives of the three fungal enzymes varied significantly, with MetE from A. sojae having the longest (> 600 min, k_D=0), and R. delemar the shortest (17 min). Knowledge of the kinetics of these enzymes is important for understanding methionine synthesis in fungi and a first step in promoting methionine synthesis in fungally fermented plant foods.