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

Abstract

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.