The engineered dimer of L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii: The role of intersubunit interactions in IlvD/EDD family

Engineering of protein oligomeric state or quaternary structure is a promising approach to adjust proteins for utilization in different applications and research. This study provides a detailed investigation of oligomerization within IlvD/EDD superfamily. A large 255 kDa tetrameric protein of the IlvD/EDD superfamily, L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii, was engineered to create a dimeric form containing the [2Fe-2S] catalytic center. Four variants, which contain mutations at two locations of the dimer-dimer interface, were produced. The mutants were analyzed using UV-vis and native mass spectrometry, while iron composition and enzymatic activity with five substrates were also investigated. The three variants with mutations closer to the catalytic center showed a color change with shifted maximum absorption at 455 nm and had reduced catalytic activity. One of the variants, D39K, located farther from the active center, maintained a similar color to the wild-type enzyme. The D39K had a mild decline in the catalytic activity, and it existed in dimeric form. The three-dimensional structure analysis suggests that the D39K mutation disrupted a larger ionic-interaction network on the protein surface, thus preventing the formation of the tetramer.