Structural and functional similarities in the ADP-forming amide bond ligase superfamily: Implications for a substrate-induced conformational change in folylpolyglutamate synthetase

Comparison of the three-dimensional structures of folylpolyglutamate synthe tase (FPGS) and the bacterial cell wall Ligase UDP-N-acetylmuramoyl-L-alani ne:D-glutamate ligase (MurD) reveals that these two enzymes have a remarkab le structural similarity despite a low level of sequence identity. Both enz ymes have a modular, multi-domain structure and catalyse a similar Am-depen dent reaction involving the addition of a glutamate residue to a carboxylat e-containing substrate, tetrahydrofolate in the case of FPGS, and UDP-N-ace tylmuramoyl-L-alanine in the case of MurD. Site-directed mutations of selec ted residues in the active site of Lactobacillus casei FPGS (P74A, E143A, E 143D, E143Q, K185A, D313A, H316A, G411A and S412A) showed that most of thes e changes resulted in an almost complete loss of activity. Several of these amino acid residues in FPGS are found in structurally equivalent positions to active-site residues in MurD. Some insights into the function of these residues in FPGS activity are proposed, based on the roles surmised from th e structures of two MurD UDP-N-acetylmuramoyl-L-alanine ADP complexes and a MurD UDP-N-acetylmuramoyl-L-alanine-D-glutamate complex. Furthermore, the comparison has led us to propose that conformational changes induced by sub strate binding in the reaction mechanism of FPGS result in a movement of th e domains towards each other to more closely resemble the orientation of th e corresponding domains in MurD. This relative domain movement may be a key feature of this new family of ADP-forming amide bond ligases. (C) 2000 Aca demic Press.