Comparative folding analyses of unknotted versus trefoil-knotted ornithine transcarbamylases suggest stabilizing effects of protein knots.

Ornithine transcarbamylases (OTCs) are conserved enzymes involved in arginine biosynthesis in microbes and the urea cycle in mammals. Recent bioinformatics analyses identified two unique OTC variants, N-succinyl-l-ornithine transcarbamylase from Bacteroides fragilis (BfSOTC) and N-acetyl-l-ornithine transcarbamylase from Xanthomonas campestris (XcAOTC). These two variants diverged from other OTCs during evolution despite sharing the common tertiary and quaternary structures, with the exception that the substrate recognition motifs are topologically knotted. The OTC family therefore offers a unique opportunity for investigating the importance of protein knots in biological functions and folding stabilities. Using hydrogen-deuterium exchange-coupled mass spectrometry, we compared the native dynamics of BfSOTC and XcAOTC with respect to the unknotted ornithine transcarbamylase from Escherichia coli (EcOTC). Our results suggest that, in addition to substrate specificity, the knotted structures in XcAOTC and BfSOTC may play an important role in stabilizing the folding dynamics, particularly around the knotted structural elements.