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 (E), 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, of the three fungal MetE enzymes on the methyl donor (6S)-5-methyl-tetrahydropteroyl-L-glutamate ranged from 1.2 to 3.3 min and K values varied from 0.8 to 6.8 µM. The k was lowest for the R. delemar MetE, but this enzyme also had the lowest K thus resulting in the highest k/K of ∼1.4 min µM among the three fungal enzymes. The k was higher for the E. coli enzyme, 12 min, but K was 6.4 µM, resulting in k/K of ∼1.9 min µM. The E values of the fungal MetEs ranged from 52 to 97 kJ mole and were higher than that of the E. coli MetE (38.7 kJ mole ). The predicted structural folds of the MetEs were very similar. T 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=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.