Functional assessment of surface loops: deletion of eukaryote-specific peptide inserts in thymidylate synthase of Saccharomyces cerevisiae

The primary structure is known for at least 29 thymidylate synthases and th e crystal structure is known for several from both prokaryotes and eukaryot es. All these are markedly similar making thymidylate synthase one of the m ost highly conserved enzymes known. There are, however, two surface loops, one near the active site and the other near the dimer interface, which exis t in distinctly prokaryotic and eukaryotic versions. Specifically, in eukar yotes these two surface loops have small peptide inserts conserved in size and partly conserved in sequence, that are not present in the prokaryotic t hymidylate synthases. To address the possibility that these inserts provide eukaryote-specific functions the Saccharomyces cerevisiae loops were indiv idually modified to mimic their prokaryotic counterparts. Altering the surf ace loop near the active site increased K-m for the nucleotide substrate an d decreased apparent V-max. Mutant variants with alterations in the other s urface loop were unable to dimerize, Therefore these surface loops have acq uired, perhaps by way of the eukaryotic inserts, characteristics that are i mportant for catalytic activity and quaternary structure respectively. (C) 1999 Elsevier Science B.V. All rights reserved.